Methods of Treating a Tauopathy

ABSTRACT

The present disclosure provides methods of treating a tauopathy, involving administering an anti-Tau antibody. The present disclosure also provides anti-Tau antibodies, and formulations comprising same, for use in the methods.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication Nos. 61/683,902, filed Aug. 16, 2012, 61/754,085, filed Jan.18, 2013, 61/781,823, filed Mar. 14, 2013, 61/813,797, filed Apr. 19,2013, and 61/833,355, filed Jun. 10, 2013, each of which applications isincorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “IPRN-745WOSeqList_ST25.txt” created on Aug. 15, 2013 and having a size of 69 KB.The contents of the text file are incorporated by reference herein intheir entirety.

INTRODUCTION

The microtubule associated protein tau is abundant in the centralnervous system and is produced primarily by neurons. The primaryfunction of tau is to stabilize microtubules. Six tau isoforms exist inthe adult human brain; tau isoforms are the products of alternativesplicing of a single gene.

Tauopathies are a class of neurodegenerative diseases resulting from thepathological aggregation of tau protein in so-called neurofibrillarytangles (NFT) in the brain. Some examples of tauopathies includefrontotemporal dementia (FTD), Alzheimer's disease, progressivesupranuclear palsy, corticobasal degeneration, and frontotemporal lobardegeneration.

There is a need in the art for methods of treating tauopathies, and forreagents suitable for use in such methods.

SUMMARY

The present disclosure provides methods of treating a tauopathy,involving administering an anti-Tau antibody. The present disclosurealso provides anti-Tau antibodies, and formulations comprising same, foruse in the methods.

FEATURES

The present disclosure provides an isolated humanized monoclonalantibody that specifically binds an epitope within amino acids 15-24 ofa Tau polypeptide. In some cases, the epitope does not comprise aphosphorylated amino acid. In some cases, the epitope does not comprisea nitrated amino acid. In some instances, the epitope comprises aphosphorylated amino acid, a nitrated amino acid, or both aphosphorylated amino acid and a nitrated amino acid.

The present disclosure provides an isolated antibody comprising ahumanized light chain framework region; and a humanized heavy chainframework region, wherein the isolated antibody competes for binding toan epitope in an N-terminal region of a Tau polypeptide with an antibodythat comprises: a) a light chain region comprising: i) a V_(L) CDR1comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) aV_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8; and (iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H)CDR1 comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10;(ii) a V_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 orSEQ ID NO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequenceof SEQ ID NO:6 or SEQ ID NO:12. In some cases, the light chain regionand the heavy chain region are present in separate polypeptides. In somecases, the light chain region and the heavy chain region are present ina single polypeptide. In some cases, the heavy chain region is of theisotype IgG1, IgG2, IgG3, or IgG4. In some cases, the heavy chain regionis of the isotype IgG4. In some of these embodiments, the hinge regioncomprises an S241P substitution. See, e.g., Angal et al. (1993)Mol.Immunol. 30:105. In some cases, the antibody is a Fv, scFv, Fab,F(ab′)2, or Fab′. In some cases, the antibody comprises a covalentlylinked non-peptide synthetic polymer, e.g., a poly(ethylene glycol)polymer. In some cases, the antibody is fused, directly or via a linker,to a carrier molecule, a peptide or a protein that promotes the crossingof the blood-brain barrier. In some cases, the epitope is within aminoacids 15-24 of a Tau polypeptide. In some cases, the humanized lightchain framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of theamino acid substitutions depicted in Table 3. In some instances, thehumanized heavy chain framework region comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 of the amino acid substitutions depicted in Table 2.

The present disclosure provides an isolated antibody, wherein theantibody is a Fv, scFv, Fab, F(ab′)2, or Fab′, and wherein the antibodycompetes for binding to an epitope in an N-terminal region of a Taupolypeptide with an antibody that comprises: a) a light chain regioncomprising: i) a V_(L) CDR1 comprising an amino acid sequence of SEQ IDNO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising an amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:8; and (iii) a V_(L) CDR3 comprising anamino acid sequence of SEQ ID NO:3 or SEQ ID NO:9; and b) a heavy chainregion comprising: (i) a V_(H) CDR1 comprising an amino acid sequence ofSEQ ID NO:4 or SEQ ID NO:10; (ii) a V_(H) CDR2 comprising an amino acidsequence of SEQ ID NO:5 or SEQ ID NO:11; and (iii) a V_(H) CDR3comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:12. Insome cases, the isolated antibody comprises a humanized light chainframework region. In some cases, the humanized light chain frameworkregion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acidsubstitutions depicted in Table 3. In some cases, the isolated antibodycomprises a humanized heavy chain framework region. In some cases, thehumanized heavy chain framework region comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 of the amino acid substitutions depicted in Table 2.

The present disclosure provides an isolated antibody, wherein theisolated antibody comprises a human light chain constant region and ahuman heavy chain constant region, and wherein the isolated antibodycompetes for binding to an epitope in an N-terminal region of a Taupolypeptide with an antibody that comprises: a) a light chain regioncomprising: i) a V_(L) CDR1 comprising an amino acid sequence of SEQ IDNO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising an amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:8; and (iii) a V_(L) CDR3 comprising anamino acid sequence of SEQ ID NO:3 or SEQ ID NO:9; and b) a heavy chainregion comprising: (i) a V_(H) CDR1 comprising an amino acid sequence ofSEQ ID NO:4 or SEQ ID NO:10; (ii) a V_(H) CDR2 comprising an amino acidsequence of SEQ ID NO:5 or SEQ ID NO:11; and (iii) a V_(H) CDR3comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:12.

The present disclosure provides a pharmaceutical formulation comprising:a) an anti-Tau antibody of the present disclosure; and b) apharmaceutically acceptable excipient.

The present disclosure provides a pharmaceutical formulation comprising:a) an antibody that specifically binds an epitope within an N-terminalportion of Tau, wherein the antibody comprises: (i) a V_(L) CDR1comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) aV_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8; (iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:9; (iv) a V_(H) CDR1 comprising an amino acid sequenceof SEQ ID NO:4 or SEQ ID NO:10; (v) a V_(H) CDR2 comprising an aminoacid sequence of SEQ ID NO:5 or SEQ ID NO:11; and (vi) a V_(H) CDR3comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:12; and b)a pharmaceutically acceptable excipient suitable for administration to ahuman, wherein the formulation is free of endotoxins. In some cases, theantibody comprises a humanized light chain framework region. In somecases, the humanized light chain framework region comprises 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 of the amino acid substitutions depicted in Table3. In some cases, the antibody comprises a humanized heavy chainframework region. In some cases, the humanized heavy chain frameworkregion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the aminoacid substitutions depicted in Table 2. In some cases, the antibody isencapsulated in a liposome. In some cases, the antibody is formulatedwith an agent that facilitates crossing the blood-brain barrier. In somecases, the antibody is fused, directly or via a linker, to a carriermolecule, a peptide or a protein that promotes the crossing of theblood-brain barrier. In some cases, the antibody is a Fv, scFv, Fab,F(ab′)2, or Fab′.

The present disclosure provides a recombinant expression vectorcomprising a nucleotide sequence encoding an anti-Tau antibody of thepresent disclosure, wherein the nucleotide sequence is operably linkedto a transcriptional control element that is active in a eukaryoticcell. The present disclosure provides an in vitro host cell geneticallymodified with a recombinant expression vector of the present disclosure.

The present disclosure provides a sterile container comprising apharmaceutical formulation of the present disclosure. In some cases, thecontainer is a syringe.

The present disclosure provides a method of treating a tauopathy in anindividual, the method comprising administering to the individual ananti-Tau antibody of the present disclosure, or a pharmaceuticalcomposition of the present disclosure.

The present disclosure provides a method of treating a tauopathy in anindividual, the method comprising administering to the individual apharmaceutical composition comprising: a) an antibody that competes forbinding to an epitope in an N-terminal region of a Tau polypeptide withan antibody that comprises: i) light chain complementarity-determiningregions (CDRs) of an antibody depicted in FIG. 1B; and heavy chain CDRsof an antibody depicted in FIG. 1A; or ii) light chain CDRs of anantibody depicted in FIG. 2B; and heavy chain CDRs of an antibodydepicted in FIG. 2A; and b) a pharmaceutically acceptable excipientsuitable for administration to a human. In some cases, the antibodycomprises: (i) a V_(L) CDR1 comprising an amino acid sequence of SEQ IDNO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising an amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:8; (iii) a V_(L) CDR3 comprising an aminoacid sequence of SEQ ID NO:3 or SEQ ID NO:9; (iv) a V_(H) CDR1comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10; (v) aV_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 or SEQ IDNO:11; and (vi) a V_(H) CDR3 comprising an amino acid sequence of SEQ IDNO:6 or SEQ ID NO:12. In some cases, the antibody comprises a humanizedlight chain framework region. In some cases, the antibody comprises ahumanized heavy chain framework region. In some cases, the antibody isencapsulated in a liposome. In some cases, the antibody is formulatedwith an agent that facilitates crossing the blood-brain barrier. In somecases, the antibody is fused, directly or via a linker, to a carriermolecule, a peptide or a protein that promotes the crossing of theblood-brain barrier. In some cases, the antibody is a Fv, scFv, Fab,F(ab′)2, or Fab′. In some cases, the administering is intravenous. Insome cases, the administering is intrathecal.

In some cases, administration of a subject anti-Tau antibody results ina change in one or more of: a) the amount of free extracellular tau inbrain tissue; b) the amount of free extracellular tau in interstitialfluid (ISF); c) the amount of free extracellular tau in cerebrospinalfluid (CSF); d) the neuron-to-neuron spread of tau; e) the amount ofintraneuron tau aggregates; f) the degree of microglial and/or astrocyteactivation; g) the amount of phosphorylated or hyperphosphorylated tau;h) the amount of total Tau or free tau in ISF or CSF; i) the amount ofintracellular N-terminal tau fragments; j) neuronal hyperactivity; k)the amount of Aβ40 and/or Aβ42 in CSF; 1) the Aβ plaque burden; m)secretion of Aβ40 and/or Aβ42 from a neuron; n) amyloid precursorprotein (APP) promoter activity; o) APP mRNA and/or protein level; p)the activity of beta-secretase and/or gamma secretase; q) the activationstate of an Aβ induced signaling pathway; r) the amount of intracellulartotal tau or free tau; s) the amount of anti-tau antibody-bound tau inISF or CSF; and t) the amount of intracellular anti-Tau antibody-boundtau.

In some cases, a method of the present disclosure for treating atauopathy in an individual further comprises administering at least oneadditional agent that treats the tauopathy.

The present disclosure provides a method of monitoring progression of atauopathy in an individual, the method comprising: a) determining afirst level of a Tau polypeptide in a biological sample obtained fromthe individual at a first time point; b) determining a second level of aTau polypeptide in a biological sample obtained from the individual at asecond time point; and c) comparing the second level of Tau with thefirst level of Tau, wherein said determining comprises: i) contactingthe biological sample with an antibody of any one of claims 1, 5, 16,and 21; and ii) quantitating binding of the antibody to Tau polypeptidepresent in the sample. In some cases, the biological sample iscerebrospinal fluid, blood, plasma, serum, urine, or saliva. In somecases, the quantitated Tau polypeptide is total Tau polypeptide. In somecases, the quantitated Tau polypeptide is an N-terminal fragment of afull-length Tau polypeptide. In some cases, the first time point is atime point before initiation of a treatment regimen, and the second timepoint is a time point after initiation of a treatment regimen.

The present disclosure provides a method of detecting a Tau polypeptidein a living individual in vivo, the method comprising: a) administeringto the individual an antibody of any one of claims 1, 5, 16, and 21; andb) detecting binding of the antibody to tau polypeptide in a braintissue in the individual using an imaging method. In some cases, theantibody comprises a contrast agent suitable for use in the imagingmethod. In some cases, the imaging method is magnetic resonance imagingor positron emission tomography.

The present disclosure provides an in vitro method of detecting a Taupolypeptide in a biological sample obtained from an individual, themethod comprising: a) contacting the biological sample with an antibodycompetes for binding to an epitope within the N-terminal region of Tauwith an antibody that comprises: i) light chaincomplementarity-determining regions (CDRs) of an antibody depicted inFIG. 1B; and heavy chain CDRs of an antibody depicted in FIG. 1A; or ii)light chain CDRs of an antibody depicted in FIG. 2B; and heavy chainCDRs of an antibody depicted in FIG. 2A; and b) detecting binding of theantibody to Tau polypeptide present in the sample. In some cases, thebiological sample is blood, serum, plasma, urine, saliva, orcerebrospinal fluid. In some cases, the individual is suspected ofhaving a tauopathy, has been diagnosed as having a tauopathy, or has agenetic predisposition to developing a tauopathy. In some cases, themethod is quantitative. In some cases, the Tau polypeptide detected istotal Tau polypeptide. In some cases, the Tau polypeptide detected is anN-terminal fragment of a full-length Tau polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide amino acid sequences of IPN001 VH (FIG. 1A) andVL (FIG. 1B). Complementarity-determining regions (CDRs) are in boldtext and underlined.

FIGS. 2A and 2B provide amino acid sequences of IPN002 VH (FIG. 2A) andVL (FIG. 2B). Complementarity-determining regions (CDRs) are in boldtext and underlined.

FIGS. 3A-D depicts the effect of anti-Tau antibody IPN002 onTau-mediated membrane depolarization in cortical neurons.

FIGS. 4A-C depict affinity isolation of Tau from cerebrospinal fluid(CSF).

FIG. 5 depicts quantification of CSF and conditioned medium (CM) samplespre- and post-Tau affinity isolation.

FIGS. 6A-D provide amino acid sequences of full-length human Tau.

FIG. 7 depicts detection of Tau fragments in conditioned medium, ininterstitial fluid (ISF) from P301L tau mice, and in CSF from PSP and ADpatients

FIGS. 8A-D depict induction of cortical neuron hyperactivity by anextracellular tau (eTau) fragment (FIGS. 8A-C); and reduction ofeTau-induced neuronal hyperactivity by anti-Tau antibody IPN001.

FIG. 9 depicts an amino acid sequence of humanized IPN002 VH variant 1;and a nucleotide sequence encoding the amino acid sequence.

FIG. 10 depicts an amino acid sequence of humanized IPN002 VH variant 2;and a nucleotide sequence encoding the amino acid sequence.

FIG. 11 depicts an amino acid sequence of humanized IPN002 VH variant 3;and a nucleotide sequence encoding the amino acid sequence.

FIG. 12 depicts an amino acid sequence of humanized IPN002 VH variant 4;and a nucleotide sequence encoding the amino acid sequence.

FIG. 13 depicts an amino acid sequence of humanized IPN002 Vκ variant 1;and a nucleotide sequence encoding the amino acid sequence.

FIG. 14 depicts an amino acid sequence of humanized IPN002 Vκ variant 2;and a nucleotide sequence encoding the amino acid sequence.

FIG. 15 depicts an amino acid sequence of humanized IPN002 Vκ variant 3;and a nucleotide sequence encoding the amino acid sequence.

FIG. 16 depicts an amino acid sequence of humanized IPN002 Vκ variant 4;and a nucleotide sequence encoding the amino acid sequence.

FIG. 17 provides Table 4, which shows binding properties of humanizedIPN-002 variants to eTau proteins.

FIG. 18 provides Table 5, which shows binding properties of humanizedIPN-002 variants to Tau-383.

FIGS. 19A and 19B depict properties of humanized IPN002 variants. FIG.19A depicts binding of humanized IPN-002 variants to tau present iniPSC-CN conditioned media; iPSC-CN lysates; AD brain lysates; and P301Ltau mouse brain cortex lysates; and cynomologus monkey brain lysates.FIG. 19B depicts inhibition of eTau-induced neuronal hyperactivity byhumanized IPN002 variants.

FIG. 20 depicts amino acid sequences of eTau fragments, in alignmentwith a fetal tau amino acid sequence.

FIGS. 21A-C depict proliferation responses to a humanized anti-Tauantibody (FIG. 21A), a chimeric antibody (FIG. 21B), and humanized A33(FIG. 21C).

FIG. 22 depicts the effect of IPN002 on phosphorylated Tau levels invivo.

FIG. 23 depicts reduction in free tau levels and in total tau levels ininterstitial fluid (ISF) following treatment with IPN002.

FIG. 24 depicts reduction in free tau levels in cerebrospinal fluid(CSF) following treatment with IPN002.

FIG. 25 depicts reduction of eTau-induced neuronal hyperactivity byIPN002.

FIG. 26 depicts the presence of Tau fragments in CSF from individualswith likely chronic traumatic encephalopathy.

FIG. 27 depicts binding of a humanized variant of IPN002 to synthetictau peptides using a solid phase assay.

FIG. 28 depicts binding of a humanized variant of IPN002 to synthetictau peptides using a solution phase assay.

FIG. 29 depicts binding of a humanized variant of IPN002 to recombinantTau and to a PAD peptide.

FIG. 30 depicts competition of non-biotinylated forms of synthetic taupeptides with biotinylated forms of synthetic tau peptides for bindingto a humanized variant of IPN002.

FIG. 31 depicts the effect of administration of control IgG, PHF1, orIPN002 on clasping scores in the P310L mouse model.

FIG. 32 depicts the effect of administration of control IgG, PHF1, orIPN002 on average latency in the beam walk test in the P310L mousemodel.

FIG. 33 depicts the effect of administration of control IgG, PHF1, orIPN002 on the level of free tau (tau not bound to anti-tau antibody) inCSF samples in the P310L mouse model.

FIG. 34 depicts antibody inhibition of eTau1a-induced neuronalhyperexcitability.

FIG. 35 depicts the effect of full-length, PHF1-reactive tau, or eTau1a,on neuronal hyperexcitability in cortical neurons in vitro.

FIG. 36 depicts graphically the effect of full-length, PHF1-reactivetau, or eTau1a, on neuronal hyperexcitability in cortical neurons invitro.

FIG. 37 depicts the effect of control IgG, BACE inhibitor, or IPN002 onlevels of Aβ40 (left panel) or Aβ42 (right panel) secreted from corticalneurons.

FIG. 38 depicts the effect of control IgG, BACE inhibitor, or anti-Tauantibodies on levels of Aβ40 secreted from primary cortical neurons.

FIG. 39 depicts the effect of control IgG, BACE inhibitor, or anti-Tauantibodies on levels of Aβ42 secreted from primary cortical neurons.

FIG. 40 depicts results of epitope mapping of a humanized variant ofIPN002 (hu-IPN002).

FIG. 41 depicts an assay for detecting various tau polypeptides in CSF.

FIG. 42 depicts binding of Tau in CSF by IPN002, PHF1, or a polyclonalantibody that binds a linear epitope in the C-terminal portion of Tau(pAb-tau linear epitope).

FIG. 43 depicts the effect of treatment of P301L mice with control IgG,PHF1, or IPN002 on the total tau levels in CSF.

FIGS. 44A-H depict the effect of treatment of P301L mice with controlIgG, PHF1, or IPN002 on AT8 phospho Tau in various brain regions andtissues.

FIGS. 45A-E depict the effect of treatment of P301L mice with controlIgG, PHF1, or IPN002 on the levels of phosphorylated Tau in variousbrain regions and tissues.

FIG. 46 depicts the effect of treatment of P301L mice with control IgG,PHF1, or IPN002 on the level of AT8 phospho Tau histology in thehindbrain.

FIG. 47 depicts the effect of treatment of P301L mice with control IgG,PHF1, or IPN002 on the level of AT100 phospho Tau histology in thehindbrain.

FIGS. 48A and 48B depict the effect of treatment of P301L mice withcontrol IgG, PHF1, or IPN002 on the level of GFAP protein in hippocampalhomogenate and in cortex homogenate.

FIGS. 49A and 49B depict the effect of treatment of P301L mice withcontrol IgG, PHF1, or IPN002 on the level of Iba1 protein in hippocampalhomogenate and in cortex homogenate.

FIGS. 50A and 50B depict the effect of treatment of P301L mice withcontrol IgG, PHF1, or IPN002 on the level of Aβ40 in cortex homogenateand cortex S1 fraction.

FIG. 51 depicts the effect of treatment of P301L mice with control IgG,PHF1, or IPN002 on the percent of mice able to perform in the beam walktest.

FIG. 52 depicts binding of hu-IPN002 to various Tau peptides.

FIG. 53 depicts binding of various biotinylated Tau peptides tohu-IPN002.

FIGS. 54A and 54B are schematic depictions of assays for Tau not boundto IPN002 (Free Tau) (FIG. 54A); and Tau bound to IPN002 (Bound Tau)(FIG. 54B).

DEFINITIONS

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, bi-specific antibodies, and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein. The antibodies may be detectably labeled, e.g., with aradioisotope, an enzyme which generates a detectable product, afluorescent protein, and the like. The antibodies may be furtherconjugated to other moieties, such as members of specific binding pairs,e.g., biotin (member of biotin-avidin specific binding pair), and thelike. The antibodies may also be bound to a solid support, including,but not limited to, polystyrene plates or beads, and the like. Alsoencompassed by the term are Fab′, Fv, F(ab′)₂, and or other antibodyfragments that retain specific binding to antigen, and monoclonalantibodies. An antibody may be monovalent or bivalent.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion comprises amino acid sequences ofhuman origin. For example, the humanized antibody can comprise portionsderived from an immunoglobulin of nonhuman origin with the requisitespecificity, such as a mouse, and from immunoglobulin sequences of humanorigin (e.g., chimeric immunoglobulin), joined together chemically byconventional techniques (e.g., synthetic) or prepared as a contiguouspolypeptide using genetic engineering techniques (e.g., DNA encoding theprotein portions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanizedimmunoglobulin is an immunoglobulin containing one or moreimmunoglobulin chains comprising a CDR derived from an antibody ofnonhuman origin and a framework region derived from a light and/or heavychain of human origin (e.g., CDR-grafted antibodies with or withoutframework changes). Chimeric or CDR-grafted single chain antibodies arealso encompassed by the term humanized immunoglobulin. See, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E.A. et al., European Patent Application No. 0,519,596 A1. See also,Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786;and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding singlechain antibodies.

For example, humanized immunoglobulins can be produced using syntheticand/or recombinant nucleic acids to prepare genes (e.g., cDNA) encodingthe desired humanized chain. For example, nucleic acid (e.g., DNA)sequences coding for humanized variable regions can be constructed usingPCR mutagenesis methods to alter DNA sequences encoding a human orhumanized chain, such as a DNA template from a previously humanizedvariable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17:5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.For example, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH₁) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH₁ domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains,which enables the sFv to form the desired structure for antigen binding.For a review of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)—V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents (e.g., an antibody and anantigen) and is expressed as a dissociation constant (Kd). Affinity canbe at least 1-fold greater, at least 2-fold greater, at least 3-foldgreater, at least 4-fold greater, at least 5-fold greater, at least6-fold greater, at least 7-fold greater, at least 8-fold greater, atleast 9-fold greater, at least 10-fold greater, at least 20-foldgreater, at least 30-fold greater, at least 40-fold greater, at least50-fold greater, at least 60-fold greater, at least 70-fold greater, atleast 80-fold greater, at least 90-fold greater, at least 100-foldgreater, or at least 1000-fold greater, or more, than the affinity of anantibody for unrelated amino acid sequences. Affinity of an antibody toa target protein can be, for example, from about 100 nanomolar (nM) toabout 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about100 nM to about 1 femtomolar (fM) or more. As used herein, the term“avidity” refers to the resistance of a complex of two or more agents todissociation after dilution. The terms “immunoreactive” and“preferentially binds” are used interchangeably herein with respect toantibodies and/or antigen-binding fragments.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. A subject anti-Tau antibody bindsspecifically to an epitope within a Tau polypeptide. Non-specificbinding would refer to binding with an affinity of less than about 10⁻⁷M, e.g., binding with an affinity of 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, etc.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of proteins of immunological interest” (1991); by Chothia etal., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol.Biol. 262:732-745 (1996), where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or grafted antibodies or variants thereof is intended to bewithin the scope of the term as defined and used herein. The amino acidresidues which encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison.

TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally a discontinuous amino acidsequence between about 100-120 amino acids in length but is intended toreference only those amino acids outside of the CDRs. As used herein,the term “framework region” is intended to mean each domain of theframework that is separated by the CDRs.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 90%, greater than 95%, or greater than 98%,by weight of antibody as determined by the Lowry method, for example,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornonreducing conditions using Coomassie blue or silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. In some instances, isolated antibody will be prepared by atleast one purification step.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

A “therapeutically effective amount” or “efficacious amount” refers tothe amount of an anti-Tau antibody that, when administered to a mammalor other subject for treating a disease, is sufficient to effect suchtreatment for the disease. The “therapeutically effective amount” willvary depending on the anti-Tau antibody, the disease and its severityand the age, weight, etc., of the subject to be treated.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The term“biological sample” includes urine, saliva, cerebrospinal fluid, bloodfractions such as plasma and serum, and the like.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahumanized anti-Tau antibody” includes a plurality of such antibodies andreference to “the tauopathy” includes reference to one or moretauopathies and equivalents thereof known to those skilled in the art,and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides methods of treating a tauopathy,involving administering an anti-Tau antibody. The present disclosurealso provides anti-Tau antibodies, and formulations comprising same, foruse in the methods. The present disclosure further provides in vitro andin vivo detection methods using an anti-Tau antibody described herein.

Methods of Treating a Tauopathy

The present disclosure provides methods of treating a tauopathy. Themethods generally involve administering an effective amount of ananti-Tau antibody of the present disclosure to an individual in needthereof. In some cases, administration of a subject anti-tau antibodyreduces the level of a pathological tau polypeptide in a cell, a tissue,or a fluid of an individual, and treats the tauopathy.

For example, in some embodiments, a subject method can compriseadministering to an individual in need thereof an effective amount of anisolated humanized monoclonal antibody that specifically binds anepitope within amino acids 15-24 of a Tau polypeptide. In someembodiments, the antibody is present in a pharmaceutical formulationwith a pharmaceutically acceptable excipient, e.g., a pharmaceuticallyacceptable excipient that is suitable for administration to a human.

For example, in some embodiments, a subject method can compriseadministering to an individual in need thereof an effective amount of anisolated antibody comprising a humanized light chain framework region;and a humanized heavy chain framework region, wherein the isolatedantibody competes for binding to an epitope in an N-terminal region of aTau polypeptide with an antibody that comprises: a) a light chain regioncomprising: i) a V_(L) CDR1 comprising an amino acid sequence of SEQ IDNO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising an amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:8; and (iii) a V_(L) CDR3 comprising anamino acid sequence of SEQ ID NO:3 or SEQ ID NO:9; and b) a heavy chainregion comprising: (i) a V_(H) CDR1 comprising an amino acid sequence ofSEQ ID NO:4 or SEQ ID NO:10; (ii) a V_(H) CDR2 comprising an amino acidsequence of SEQ ID NO:5 or SEQ ID NO:11; and (iii) a V_(H) CDR3comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:12. Insome embodiments, the antibody is present in a pharmaceuticalformulation with a pharmaceutically acceptable excipient, e.g., apharmaceutically acceptable excipient that is suitable foradministration to a human.

For example, in some embodiments, a subject method can compriseadministering to an individual in need thereof an effective amount of anisolated antibody, wherein the antibody is a Fv, scFv, Fab, F(ab′)2, orFab′, and wherein the antibody competes for binding to an epitope in anN-terminal region of a Tau polypeptide with an antibody that comprises:a) a light chain region comprising: i) a V_(L) CDR1 comprising an aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8; and(iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ ID NO:3 orSEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H) CDR1comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10; (ii) aV_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 or SEQ IDNO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequence of SEQID NO:6 or SEQ ID NO:12. In some embodiments, the antibody is present ina pharmaceutical formulation with a pharmaceutically acceptableexcipient, e.g., a pharmaceutically acceptable excipient that issuitable for administration to a human.

For example, in some embodiments, a subject method can compriseadministering to an individual in need thereof an effective amount of anisolated antibody, wherein the isolated antibody comprises a human lightchain constant region and a human heavy chain constant region, andwherein the isolated antibody competes for binding to an epitope in anN-terminal region of a Tau polypeptide with an antibody that comprises:a) a light chain region comprising: i) a V_(L) CDR1 comprising an aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8; and(iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ ID NO:3 orSEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H) CDR1comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10; (ii) aV_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 or SEQ IDNO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequence of SEQID NO:6 or SEQ ID NO:12. In some embodiments, the antibody is present ina pharmaceutical formulation with a pharmaceutically acceptableexcipient, e.g., a pharmaceutically acceptable excipient that issuitable for administration to a human.

An anti-Tau antibody of the present disclosure binds extracellular tau.“Extracellular tau” (“eTau”), as used herein, encompasses any Taupolypeptide that can be detected in cerebrospinal fluid (CSF) orinterstitial fluid (ISF). In some embodiments, eTau is a polypeptidehaving a length of 175 amino acids and comprising amino acids 2-176 offull-length tau; for example, in some embodiments eTau is a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:45. In someembodiments, eTau is a polypeptide having a length of 171 amino acidsand comprising amino acids 2-172 (SEQ ID NO:44) of full-length tau; forexample, in some embodiments eTau is a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:44. In some embodiments, eTau is aneTau-2 polypeptide comprising the amino acid sequence set forth in SEQID NO:46. In some embodiments, eTau is an eTau-3 polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO:47. In some embodiments,eTau is an eTau-4 polypeptide comprising the amino acid sequence setforth in SEQ ID NO:48.

In some cases, an eTau polypeptide has a length of from about 50 aminoacids to about 175 amino acids, e.g., from about 50 amino acids (aa) toabout 75 aa, from about 75 aa to about 100 aa, from about 100 aa toabout 125 aa, from about 125 aa to about 150 aa, or from about 150 aa toabout 175 aa; and can comprise from 50 to about 75, from about 75 toabout 100, from about 100 to about 125, from about 125 to about 150, orfrom about 150 to about 175, contiguous amino acids of amino acids 2-176of full-length tau. Exemplary eTau polypeptides are depicted in FIG. 20.

As described in more detail below, an anti-Tau antibody of the presentdisclosure specifically binds Tau, where the epitope bound by theantibody is a linear epitope, and comprises amino acid residues withinan amino-terminal (N-terminal) portion of Tau, e.g., within amino acids1-25 of Tau, within amino acids 1-18 of Tau, within amino acids 9 to 18of Tau, within amino acids 13-24 of Tau, within amino acids 15-44 ofTau, or within amino acids 15-24 of Tau. Amino acid sequences of humanTau isoforms are depicted in FIGS. 6A-D. Amino acids 1-18 of Tau are:MAEPRQEFEVMEDHAGTY; SEQ ID NO:53). See, e.g., Garcia-Sierra et al.(2003) J. Alzheimer's Disease 5:65; and Horowitz et al. (2004) J.Neurosci. 24:7895. Amino acids 15-24 of Tau are: AGTYGLGDRK (SEQ IDNO:51).

In some embodiments, an anti-Tau antibody of the present disclosurespecifically binds Tau, where the epitope bound by the antibody is alinear epitope, and comprises amino acid residues within amino acids1-25 of Tau, within amino acids 13-24 of Tau, or within amino acids15-24 of Tau. In some cases, a humanized anti-Tau antibody of thepresent disclosure specifically binds a linear epitope comprising aminoacid residues within amino acids 1-25 of Tau, within amino acids 13-24of Tau, or within amino acids 15-24 of Tau, where the epitope does notinclude a phosphorylated amino acid. In some cases, a humanized anti-Tauantibody of the present disclosure specifically binds a linear epitopecomprising amino acid residues within amino acids 1-25 of Tau, withinamino acids 13-24 of Tau, or within amino acids 15-24 of Tau, where theepitope includes a phosphorylated amino acid. In some cases, a humanizedanti-Tau antibody of the present disclosure specifically binds a linearepitope comprising amino acid residues within amino acids 1-25 of Tau,within amino acids 13-24 of Tau, or within amino acids 15-24 of Tau,where the epitope does not include a nitrated amino acid. In some cases,a humanized anti-Tau antibody of the present disclosure specificallybinds a linear epitope comprising amino acid residues within amino acids1-25 of Tau, within amino acids 13-24 of Tau, or within amino acids15-24 of Tau, where the epitope includes a nitrated amino acid. In somecases, a humanized anti-Tau antibody of the present disclosurespecifically binds a linear epitope comprising amino acid residueswithin amino acids 1-25 of Tau, within amino acids 13-24 of Tau, orwithin amino acids 15-24 of Tau, where the epitope includes a nitratedamino acid, and does not include a phosphorylated amino acid. In somecases, a humanized anti-Tau antibody of the present disclosurespecifically binds a linear epitope comprising amino acid residueswithin amino acids 1-25 of Tau, within amino acids 13-24 of Tau, orwithin amino acids 15-24 of Tau, where the epitope includes aphosphorylated amino acid and does not include a nitrated amino acid. Insome cases, a humanized anti-Tau antibody of the present disclosurespecifically binds a linear epitope comprising amino acid residueswithin amino acids 1-25 of Tau, within amino acids 13-24 of Tau, orwithin amino acids 15-24 of Tau, where the epitope includes a nitratedamino acid and a phosphorylated amino acid.

For example, in some embodiments, an anti-Tau antibody of the presentdisclosure specifically binds a linear epitope comprising amino acidresidues within amino acids AGTYGLGDRK (SEQ ID NO:51) of Tau. In someembodiments, a humanized anti-Tau antibody of the present disclosurespecifically binds a linear epitope comprising amino acid residueswithin amino acids AGTYGLGDRK (SEQ ID NO:51) of Tau.

In some cases, a humanized anti-Tau antibody of the present disclosurespecifically binds a linear epitope comprising amino acid residueswithin amino acids AGTYGLGDRK (SEQ ID NO:51) of Tau, where the epitopedoes not include a phosphorylated amino acid. In some cases, a humanizedanti-Tau antibody of the present disclosure specifically binds a linearepitope comprising amino acid residues within amino acids AGTYGLGDRK(SEQ ID NO:51) of Tau, where the epitope includes a phosphorylated aminoacid. In some cases, a humanized anti-Tau antibody of the presentdisclosure specifically binds a linear epitope comprising amino acidresidues within amino acids AGTYGLGDRK (SEQ ID NO:51) of Tau, where theepitope does not include a nitrated amino acid. In some cases, ahumanized anti-Tau antibody of the present disclosure specifically bindsa linear epitope comprising amino acid residues within amino acidsAGTYGLGDRK (SEQ ID NO:51) of Tau, where the epitope includes a nitratedamino acid. In some cases, a humanized anti-Tau antibody of the presentdisclosure specifically binds a linear epitope comprising amino acidresidues within amino acids AGTYGLGDRK (SEQ ID NO:51) of Tau, where theepitope includes a nitrated amino acid, and does not include aphosphorylated amino acid. In some cases, a humanized anti-Tau antibodyof the present disclosure specifically binds a linear epitope comprisingamino acid residues within amino acids AGTYGLGDRK (SEQ ID NO:51) of Tau,where the epitope includes a phosphorylated amino acid and does notinclude a nitrated amino acid. In some cases, a humanized anti-Tauantibody of the present disclosure specifically binds a linear epitopecomprising amino acid residues within amino acids AGTYGLGDRK (SEQ IDNO:51) of Tau, where the epitope includes a nitrated amino acid and aphosphorylated amino acid.

In some cases, a method of the present disclosure for treating atauopathy comprises administering to an individual in need thereof apharmaceutical composition comprising: a) an anti-Tau antibodycomprising: i) one, two, or three light chaincomplementarity-determining regions (CDRs) of an antibody depicted inFIG. 1; and one, two, or three heavy chain CDRs of an antibody depictedin FIG. 1; or ii) one, two, or three light chain CDRs of an antibodydepicted in FIG. 2; and one, two, or three heavy chain CDRs of anantibody depicted in FIG. 2; and b) a pharmaceutically acceptableexcipient suitable for administration to a human.

In some cases, a method of the present disclosure for treating atauopathy comprises administering to an individual in need thereof apharmaceutical composition comprising: a) an antibody that specificallybinds an epitope within a human Tau polypeptide, where the antibodycompetes for binding to the epitope with an antibody that comprises: i)light chain complementarity-determining regions (CDRs) of an antibodydepicted in FIG. 1B; and heavy chain CDRs of an antibody depicted inFIG. 1A; or ii) light chain CDRs of an antibody depicted in FIG. 2B; andheavy chain CDRs of an antibody depicted in FIG. 2A; and b) apharmaceutically acceptable excipient suitable for administration to ahuman.

In some cases, a method of the present disclosure for treating atauopathy comprises administering to an individual in need thereof apharmaceutical composition comprising: a) an antibody that competes forbinding with humanized IPN002 (hu-IPN002) to an epitope in Tau that isrecognized by hu-IPN002 (e.g., a linear epitope within an N-terminalportion of Tau, e.g., within amino acids 1-25 of Tau, within amino acids1-18 of Tau, within amino acids 9 to 18 of Tau, within amino acids 15-44of Tau, within amino acids 13-24 of Tau, or within amino acids 15-24 ofTau); and b) a pharmaceutically acceptable excipient suitable foradministration to a human.

IPN001 (also referred to herein as “IPN1” or “IPN-1”) and IPN002 (alsoreferred to herein as “IPN2” or “IPN-2”) specifically bind Tau. Theepitope bound by IPN001 is a linear epitope, and comprises amino acidresidues within an amino-terminal (N-terminal) portion of Tau, e.g.,within amino acids 1-25 of Tau.

In some instances, an anti-Tau antibody of the present disclosure thatis suitable for use in a method of treating a tauopathy comprises: a) alight chain variable region comprising: i) one, two, or three V_(L) CDRsof an IPN001 antibody; and ii) a humanized light chain framework region;and b) a heavy chain variable region comprising: i) one, two, or threeV_(H) CDRs of an IPN001 antibody; and ii) a humanized heavy chainframework region; where the V_(H) and V_(L) CDRs are as defined by Kabat(see, e.g., Table 1, above; and Kabat et al., U.S. Dept. of Health andHuman Services, “Sequences of proteins of immunological interest”(1991)).

In some instances, an anti-Tau antibody of the present disclosure thatis suitable for use in a method of treating a tauopathy comprises: a) alight chain region comprising: i) one, two, or three V_(L) CDRs of anIPN001 antibody; and ii) a humanized light chain framework region; andb) a heavy chain region comprising: i) one, two, or three V_(H) CDRs ofan IPN001 antibody; and ii) a humanized heavy chain framework region;where the V_(H) and V_(L) CDRs are as defined by Chothia (see, e.g.,Table 1, above; and Chothia et al., J. Mol. Biol. 196:901-917 (1987)).

In other instances, an anti-Tau antibody of the present disclosure thatis suitable for use in a method of treating a tauopathy comprises: a) alight chain region comprising: i) one, two, or three V_(L) CDRs of anIPN002 antibody; and ii) a humanized light chain framework region; andb) a heavy chain region comprising: i) one, two, or three V_(H) CDRs ofan IPN002 antibody; and ii) a humanized heavy chain framework region;where the V_(H) and V_(L) CDRs are as defined by Kabat (see, e.g., Table1, above; and Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of proteins of immunological interest” (1991)).

In other instances, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, where the epitope is within an amino-terminal (N-terminal)portion of Tau, e.g., within amino acids 1-25 of Tau, within amino acids1-18 of Tau, within amino acids 9 to 18 of Tau, within amino acids 15-44of Tau, within amino acids 13-24 of Tau, or within amino acids 15-24 ofTau) comprises: a) a light chain region comprising: i) one, two, orthree V_(L) CDRs of an IPN002 antibody; and ii) a humanized light chainframework region; and b) a heavy chain region comprising: i) one, two,or three V_(H) CDRs of an IPN002 antibody; and ii) a humanized heavychain framework region; where the V_(H) and V_(L) CDRs are as defined byChothia (see, e.g., Table 1, above; and Chothia et al., J. Mol. Biol.196:901-917 (1987)).

In some cases, a method of the present disclosure for treating atauopathy comprises administering to an individual in need thereof aneffective amount of a pharmaceutical composition comprising: a) anantibody that specifically binds a linear epitope within anamino-terminal (N-terminal) portion of Tau, e.g., within amino acids1-25 of Tau, within amino acids 1-18 of Tau, within amino acids 9 to 18of Tau (where amino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ IDNO:53), within amino acids 15-44 of Tau, within amino acids 13-24 ofTau, or within amino acids 15-24 of Tau (where amino acids 15-24 of Tauare: AGTYGLGDRK (SEQ ID NO:51)), where the antibody comprises: (i) aV_(L) CDR1 comprising an amino acid sequence of SEQ ID NO:1 or SEQ IDNO:7; (ii) a V_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2or SEQ ID NO:8; (iii) a V_(L) CDR3 comprising an amino acid sequence ofSEQ ID NO:3 or SEQ ID NO:9; (iv) a V_(H) CDR1 comprising an amino acidsequence of SEQ ID NO:4 or SEQ ID NO:10; (v) a V_(H) CDR2 comprising anamino acid sequence of SEQ ID NO:5 or SEQ ID NO:11; and (vi) a V_(H)CDR3 comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:12;and b) a pharmaceutically acceptable excipient suitable foradministration to a human.

V_(H) and V_(L) amino acid sequences of IPN001 are depicted in FIGS. 1Aand 1B. CDRs (as defined by Kabat) are in bold text and underlined.V_(H) and V_(L) amino acid sequences of IPN002 are depicted in FIGS. 2Aand 2B. CDRs (as defined by Kabat) are in bold text and underlined.

SEQ ID NOs:1-12 are as follows:

(SEQ ID NO: 1) RSSQTILHSNGNTYLE; (SEQ ID NO: 2) KVSKRFS; (SEQ ID NO: 3)FQGSLVPWA; (SEQ ID NO: 4) SYGMS; (SEQ ID NO: 5) TISSSGSRTYFPDSVKG;(SEQ ID NO: 6) TWDGAMDY; (SEQ ID NO: 7) KSSQSIVHSNGNTYLE; (SEQ ID NO: 8)KVSNRFS; (SEQ ID NO: 9) FQGSLVPWA; (SEQ ID NO: 10) KYGMS;(SEQ ID NO: 11) TISSSGSRTYYPDSVKG; (SEQ ID NO: 12) SWDGAMDY.

In some cases, the antibody comprises a humanized light chain frameworkregion and/or a humanized heavy chain framework region. Humanizedanti-Tau antibodies are described in detail below.

A tauopathy is a disorder characterized by an abnormal level of tau in acell, a tissue, or a fluid in an individual. In some cases, a tauopathyis characterized by the presence in a cell, a tissue, or a fluid ofelevated (higher than normal) levels of tau or tau polypeptides and/orpathological forms of tau. For example, in some cases, a tauopathy ischaracterized by the presence in brain tissue and/or cerebrospinal fluidof elevated levels of tau or tau polypeptides and/or pathological formsof tau. A “higher than normal” level of tau in a cell, a tissue, or afluid indicates that the level of tau in the tissue or fluid is higherthan a normal, control level, e.g., higher than a normal, control levelfor an individual or population of individuals of the same age group.See, e.g., Blomberg et al. (2001) “Cerebrospinal fluid tau levelsincrease with age in healthy individuals” Dement. Geriatr. Cogn. Disord.12:127. In some cases, an individual having a tauopathy exhibits one ormore additional symptoms of a tauopathy (e.g., cognitive decline).

In other cases, a tauopathy is characterized by the presence in a cell,a tissue, or a fluid of lower than normal levels of tau. A “lower thannormal” level of tau in a tissue or a fluid indicates that the level oftau in the cell, tissue, or fluid is lower than a normal, control level,e.g., lower than a normal, control level for an individual or populationof individuals of the same age group.

Alzheimer's disease and certain forms of Frontotemporal dementia (Pick'sdisease, sporadic Frontotemporal dementia and Frontotemporal dementiawith Parkinsonism linked to chromosome 17) are the most common forms oftauopathy. The present disclosure provides a treatment method asdescribed above, wherein the tauopathy is Alzheimer's, Pick's disease,sporadic Frontotemporal dementia and Frontotemporal dementia withParkinsonism linked to chromosome 17. Other tauopathies include, but arenot limited to, Progressive supranuclear palsy (PSP), Corticobasaldegeneration (CBD) and Subacute sclerosing panencephalitis.

A neurodegenerative tauopathy includes Alzheimer's disease, amyotrophiclateral sclerosis/parkinsonism-dementia complex, argyrophilic graindementia, British type amyloid angiopathy, cerebral amyloid angiopathy,corticobasal degeneration, Creutzfeldt-Jakob disease, dementiapugilistica, diffuse neurofibrillary tangles with calcification, Down'ssyndrome, frontotemporal dementia (FTD), frontotemporal dementia withparkinsonism linked to chromosome 17, frontotemporal lobar degeneration,Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease,inclusion body myositis, multiple system atrophy, myotonic dystrophy,Niemann-Pick disease type C, non-Guamanian motor neuron disease withneurofibrillary tangles, Pick's disease, postencephalitic parkinsonism,prion protein cerebral amyloid angiopathy, progressive subcorticalgliosis, progressive supranuclear palsy, subacute sclerosingpanencephalitis, Tangle only dementia, multi-infarct dementia, ischemicstroke, chronic traumatic encephalopathy (CTE), traumatic brain injury(TBI), and stroke.

The present disclosure also provides methods of treating asynucleinopathy, e.g., Parkinson's disease (PD); dementia with LewyBodies (DLB); multiple system atrophy (MSA); etc. For example, PD withdementia (PDD) can be treated with a subject method.

In one embodiment, an anti-tau antibody of the present disclosureprevents or delays the onset of at least one symptom of aneurodegenerative tauopathy in a subject. In one embodiment, a subjectanti-tau antibody reduces or eliminates at least one symptom of aneurodegenerative tauopathy in a subject. The symptom may be theformation of one or more of pathological tau deposits; extracellularsoluble Tau and/or Tau fragments; hyperphosphorylated tau deposits;insoluble tau deposits; neurofibrillary tangles; neurofibrillary fibers;pre-tangle phospho-tau aggregates; intraneuronal neurofibrillarytangles; neuronal hyperactivity; and extraneuronal neurofibrillarytangles in the brain or spinal cord of a subject. The symptom may be aneurological symptom, for example, impaired cognitive function, memoryimpairment, loss of motor function, etc. In some cases, an anti-tauantibody of the present disclosure can improve cognitive function. Insome cases, an anti-tau antibody of the present disclosure can reducethe rate of decline in cognitive function. In some cases, an anti-tauantibody of the present disclosure can improve motor function. In somecases, an anti-tau antibody of the present disclosure can reduce therate of decline in motor function.

The symptom can also be the level of a Tau polypeptide in the CSF of theindividual. For example, in some embodiments, a subject anti-tauantibody, when administered in one or more doses as monotherapy or incombination therapy to an individual having a tauopathy, reduces thelevel of a Tau polypeptide in the CSF of the individual by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, or more than50%, compared to the level of the Tau polypeptide in the CSF of theindividual before treatment with the anti-tau antibody.

Administration of a subject anti-tau antibody to an individual canresult in one or more of: reduction in the amount of free extracellularTau in brain tissue; reduction in the cell-to-cell spread (e.g.,neuron-to-neuron spread) of Tau (e.g., Tau fragments); reduction in theamount of tau aggregates (e.g., intracellular (e.g., intraneuronal) tauaggregates); reduction in the amount of neurofibrillary tangles in braintissue; reduction in the level of microglial activation and/or astrocyteactivation; reduction in the amount of phosphorylated tau; reduction inthe amount of hyperphosphorylated tau; reduction in total Tau (e.g.,total intracellular Tau; and/or total extracellular Tau); reduction infree Tau (e.g., Tau that is not bound to a subject anti-Tau antibody);reduction in neuronal hyperactivity; and reduction in the amount ofN-terminal Tau fragments. “Total Tau” can include the sum total offull-length Tau of any isoform; and any N-terminal Tau fragments thatare present and that display the epitope recognized by a subjectanti-Tau antibody. Amino acid sequences of human full-length Tau arepresented in FIGS. 6A-D. Reduction in phosphorylated Tau can bedetermined using any known method, e.g., an immunological method usingan anti-phospho-Tau antibody.

Administration of a subject anti-tau antibody to an individual canresult in a change in one or more of: a) the amount of freeextracellular tau in brain tissue; b) the amount of free extracellulartau in interstitial fluid (ISF); c) the amount of free extracellular tauin cerebrospinal fluid (CSF); d) the neuron-to-neuron spread of tau; e)the amount of intraneuron tau aggregates; f) the degree of microglialand/or astrocyte activation; g) the amount of phosphorylated orhyperphosphorylated tau; h) the amount of total Tau or free tau in ISFor CSF; i) the amount of intracellular N-terminal tau fragments; j)neuronal hyperactivity; k) the amount of Aβ40 and/or Aβ42 in CSF; 1) theAβ plaque burden; m) secretion of Aβ40 and/or Aβ42 from a neuron; n)amyloid precursor protein (APP) promoter activity; o) APP mRNA and/orprotein level; p) the activity of beta-secretase and/or gamma secretase;q) the activation state of an Aβ induced signaling pathway; r) theamount of intracellular total tau or free tau; s) the amount of anti-tauantibody-bound tau in ISF or CSF; and t) the amount of intracellularanti-Tau antibody-bound tau.

Administration of a subject anti-tau antibody to an individual can insome cases improve cognitive function in the individual, or at leastreduce the rate of decline of cognitive function in the individual.

In some cases, administration of a subject anti-tau antibody to anindividual reduces the amount of free extracellular tau polypeptide(e.g., the amount of free extracellular tau polypeptide in a braintissue) by at least about 10%, at least about 20%, at least about 25%,at least about 50%, or more than 50%, compared to the amount of freeextracellular tau polypeptide in the individual before administrationwith the anti-tau antibody.

In some cases, administration of a subject anti-tau antibody to anindividual reduces the cell-to-cell (e.g., neuron-to-neuron) spread of atau polypeptide (e.g., a pathological tau polypeptide) by at least about10%, at least about 20%, at least about 25%, at least about 50%, or morethan 50%, compared to the cell-to-cell spread before administration witha subject anti-tau antibody.

In some cases, administration of a subject anti-tau antibody to anindividual reduces the amount of tau aggregates (e.g., intracellular(e.g., intraneuronal) tau aggregates) by at least about 10%, at leastabout 20%, at least about 25%, at least about 50%, or more than 50%,compared to the amount of tau aggregates before administration with theanti-tau antibody.

In some cases, administration of a subject anti-tau antibody to anindividual reduces neurotoxicity in an individual; and/or reducesneuroinflammation in an individual; and/or reduces activation ofastrocytes and microglia; and/or reduces induction of pathologicalelectrophysiological effects; and/or reduces the amount of tau inexosomes.

In some cases, administration of a subject anti-tau antibody to anindividual reduces neuronal hyperactivity by at least about 10%, atleast about 20%, at least about 25%, at least about 50%, or more than50%, compared to the level of degree of neuronal hyperactivity beforeadministration with the anti-tau antibody. In some cases, administrationof a subject anti-tau antibody to an individual reduces neuronalhyperactivity by at least about 10%, at least about 20%, at least about25%, at least about 50%, or more than 50%, as determined by whole cellpatch clamp recording of a neuron; e.g., whole cell patch clamprecording of an induced pluripotent stem cell-derived cortical neuron(iPSC-CN) or of a human cortical neuron cultures (HCC).

Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, intraperitoneal, subcutaneous, intracranial,intrathecal, intraarterial (e.g., via the carotid artery),intramuscular, intranasal, topical or intradermal administration orspinal or brain delivery. Aerosol formulations such as nasal sprayformulations include purified aqueous or other solutions of the activeagent with preservative agents and isotonic agents. Such formulationsare adjusted to a pH and isotonic state compatible with the nasal mucousmembranes.

In some cases, a subject anti-tau antibody is modified, or formulated,in such a manner as to provide the ability of the antibody to cross theblood-brain barrier. Such an antibody or antibody composition can beadministered to an individual having a tauopathy by various enteral andparenteral routes of administration including oral, intravenous, etc.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of thepresent disclosure may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

The dosage regimen will be determined by the attending physician orother medical personnel, based on various clinical factors. As is wellknown in the medical arts, dosages for any one patient depends uponvarious factors, including the patient's size, body surface area, age,the particular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. A dose of a subject anti-tau antibody can be, for example,in the range of 0.001 μg to 1000 μg; however, doses below or above thisexemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, or from about 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, or at least 1mg/kg. Doses intermediate in the above ranges are also intended to bewithin the scope of the invention. Subjects can be administered suchdoses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimensentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.

Combination Therapy

An anti-tau antibody of the present disclosure can be administered to anindividual in need thereof alone (e.g., as monotherapy); or incombination therapy with one or more additional therapeutic agents.

For the treatment of AD, suitable additional therapeutic agents include,but are not limited to, acetylcholinesterase inhibitors, including, butnot limited to, Aricept (donepezil), Exelon (rivastigmine), metrifonate,and tacrine (Cognex); an anti-Aβ antibody; non-steroidalanti-inflammatory agents, including, but not limited to, ibuprofen andindomethacin; cyclooxygenase-2 (Cox2) inhibitors such as Celebrex; andmonoamine oxidase inhibitors, such as Selegilene (Eldepryl or Deprenyl).Dosages for each of the above agents are known in the art.

Another suitable additional therapeutic agent in the treatment of AD isan agent that inhibits tau aggregation, e.g., a napthoquinone derivativethat inhibits tau aggregation, as described in U.S. Pat. No. 7,605,179.Another suitable additional therapeutic agent is an agent that inhibitsphosphorylation of tau, e.g., a 3-substituted-4-pyrimidone derivativethat inhibits tau protein kinase 1, as described in U.S. Pat. No.7,572,793.

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

Individuals to be Treated

Individuals suitable for treatment with a subject anti-tau antibodyinclude individuals who have been diagnosed as having a tauopathy;individuals at greater risk than the general population for developing atauopathy (e.g., individuals having a genetic predisposition todeveloping a tauopathy); individuals with PDD; and the like. In somecases, the individual is an adult human. In some cases, the adult humanis 30 years of age or older; 40 years of age or older, 50 years of ageor older, 60 years of age or older, 70 years of age or older, or 80years of age or older. For example, the adult human can be from 40 yearsold to 50 years old, from 50 years old to 60 years old, from 60 yearsold to 70 years old, or older than 70 years.

Methods of Reducing Aβ40 and Aβ42 Levels

The present disclosure provides a method of reducing the level of Aβ₄₀and/or Aβ₄₂ in a neuronal cell and/or extracellular fluid in anindividual. The method generally involves administering to theindividual: a) an effective amount of a humanized antibody that binds anN-terminal region of a tau polypeptide; or b) a pharmaceuticalcomposition comprising the humanized antibody.

A humanized antibody that binds an N-terminal region of a taupolypeptide, and that is suitable for use in a subject method ofreducing Aβ40 and Aβ42 in a neuronal cell and/or extracellular fluid, isa humanized antibody that binds an epitope of Tau that is within aminoacids 2-176 of Tau, e.g., within amino acids 2-15, amino acids 15-24,amino acids 24-50, amino acids 2-25, amino acids 15 to 50, amino acids50 to 75, amino acids 40 to 60, amino acids 75 to 100, amino acids 60 to80, amino acids 100 to 125, amino acids 80-115, amino acids 125 to 150,amino acids 115 to 140, amino acids 150 to 176, or amino acids 140 to160, of Tau. Exemplary Tau polypeptides are depicted in FIG. 20; anantibody that reduces the level of Aβ₄₀ and/or Aβ₄₂ in a neuronal celland/or extracellular fluid in an individual can be a humanized antibodythat specifically binds an epitope in a Tau polypeptide depicted in FIG.20.

In some cases, an antibody that reduces the level of Aβ₄₀ and/or Aβ₄₂ ina neuronal cell and/or extracellular fluid in an individual, and that issuitable for use in a subject method, is a humanized anti-Tau antibodyof the present disclosure. In some cases, the antibody is a humanizedantibody that binds an epitope within amino acids 15-24 of Tau.

Anti-Tau Antibodies

The present disclosure provides isolated anti-Tau antibodies, andpharmaceutical formulations comprising same.

The present disclosure provides an isolated antibody that specificallybinds an epitope within an N-terminal region of a Tau polypeptide (e.g.,a linear epitope within an amino-terminal (N-terminal) portion of Tau,e.g., within amino acids 1-25 of Tau, within amino acids 1-18 of Tau,within amino acids 9 to 18 of Tau (where amino acids 1-18 of Tau are:MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), within amino acids 15-44 of Tau,within amino acids 13-24 of Tau, or within amino acids 15-24 of Tau(where amino acids 15-24 of Tau are: AGTYGLGDRK (SEQ ID NO:51). In someinstances, the antibody is humanized, e.g., one or more frameworkregions of the heavy chain variable region and/or the light chainvariable region includes sequences derived from a human immunoglobulinframework.

The present disclosure provides an isolated humanized monoclonalantibody that specifically binds an epitope within amino acids 15-24 ofa Tau polypeptide. In some cases, the epitope does not comprise aphosphorylated amino acid. In some case, the epitope does not comprise anitrated amino acid. In some cases, the epitope comprises aphosphorylated amino acid, a nitrated amino acid, or both aphosphorylated amino acid and a nitrated amino acid.

Humanization of a framework region(s) reduces the risk of the antibodyeliciting a human-anti-mouse-antibody (HAMA) response in humans.Art-recognized methods of determining immune response can be performedto monitor a HAMA response in a particular patient or during clinicaltrials. Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of the therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase enzyme-linked immunosorbent assay (ELISA) analysis. In manycases, a subject humanized anti-Tau antibody does not substantiallyelicit a HAMA response in a human subject. In some cases, a subjecthumanized anti-Tau antibody has reduced immunogenic potential, asdetermined by an EpiScreen™ assay performed using CD8⁺-depletedperipheral blood mononuclear cells. In some cases, a subject humanizedanti-Tau antibody exhibits a Stimulation Index of less than 2.0.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution can be determined,in part, by computer modeling. Computer hardware and software forproducing three-dimensional images of immunoglobulin molecules are knownin the art. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, e.g., those sharing at least 60%, 70%, 80%, 90%, or morethan 90%, sequence identity or more are selected for modeling. Thesolved starting structures are modified to allow for differences betweenthe actual amino acids in the immunoglobulin chains or domains beingmodeled, and those in the starting structure. The modified structuresare then assembled into a composite immunoglobulin. Finally, the modelis refined by energy minimization and by verifying that all atoms arewithin appropriate distances from one another and that bond lengths andangles are within chemically acceptable limits.

CDR and framework regions are as defined by Kabat, Sequences of Proteinsof Immunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991). An alternative structural definition has been proposedby Chothia et al., J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989);and J. Mol. Biol. 186:651 (1989) (collectively referred to as“Chothia”). When framework residues, as defined by Kabat, supra,constitute structural loop residues as defined by Chothia, supra, theamino acids present in the mouse antibody may be selected forsubstitution into the humanized antibody. Residues which are “adjacentto a CDR region” include amino acid residues in positions immediatelyadjacent to one or more of the CDRs in the primary sequence of thehumanized immunoglobulin chain, for example, in positions immediatelyadjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia(See e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al., Science, 233:747 (1986)) and selecting these aminoacids from the donor may be desirable to keep all the antigen contactsthat provide affinity in the original antibody.

The present disclosure provides an isolated antibody comprising ahumanized light chain framework region; and a humanized heavy chainframework region, wherein the isolated antibody competes for binding toan epitope in an N-terminal region of a Tau polypeptide with an antibodythat comprises: a) a light chain region comprising: i) a V_(L) CDR1comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) aV_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8; and (iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H)CDR1 comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10;(ii) a V_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 orSEQ ID NO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequenceof SEQ ID NO:6 or SEQ ID NO:12. In some cases, the light chain regionand the heavy chain region are present in separate polypeptides. Inother cases, the light chain region and the heavy chain region arepresent in a single polypeptide. The isolated antibody can include aheavy chain that comprises a constant region of the isotype IgG1, IgG2,IgG3, or IgG4. In other cases, the antibody is a Fv, scFv, Fab, F(ab′)2,or Fab′. The antibody can comprise a covalently linked non-peptidesynthetic polymer, e.g., where the synthetic polymer is a poly(ethyleneglycol) polymer. In some cases, the isolated antibody is fused, directlyor via a linker, to a carrier molecule, a peptide or a protein thatpromotes the crossing of the blood-brain barrier. In some cases, theepitope bound by the isolated antibody is within amino acids 15-24 of aTau polypeptide. The isolated antibody humanized light chain frameworkregion can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acidsubstitutions depicted in Table 3. The isolated antibody humanized heavychain framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 of the amino acid substitutions depicted in Table 2.

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three complementarity determining regions (CDRs) of an IPN001antibody, where the CDRs are as defined by Kabat (see, e.g., Table 1,above; and Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of proteins of immunological interest” (1991)).

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three V_(L) CDRs of an IPN001 antibody; and ii) a humanizedlight chain framework region; and b) a heavy chain region comprising: i)one, two, or three V_(H) CDRs of an IPN001 antibody; and ii) a humanizedheavy chain framework region; where the V_(H) and V_(L) CDRs are asdefined by Kabat (see, e.g., Table 1, above; and Kabat et al., U.S.Dept. of Health and Human Services, “Sequences of proteins ofimmunological interest” (1991)). In some of these embodiments, theanti-Tau antibody includes a humanized V_(H) and/or V_(L) frameworkregion.

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three V_(L) CDRs of an IPN001 antibody; and ii) a humanizedlight chain framework region; and b) a heavy chain region comprising: i)one, two, or three V_(H) CDRs of an IPN001 antibody; and ii) a humanizedheavy chain framework region; where the V_(H) and V_(L) CDRs are asdefined by Chothia (see, e.g., Table 1, above; and Chothia et al., J.Mol. Biol. 196:901-917 (1987)).

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three V_(L) CDRs of an IPN002 antibody; and ii) a humanizedlight chain framework region; and b) a heavy chain region comprising: i)one, two, or three V_(H) CDRs of an IPN002 antibody; and ii) a humanizedheavy chain framework region; where the V_(H) and V_(L) CDRs are asdefined by Kabat (see, e.g., Table 1, above; and Kabat et al., U.S.Dept. of Health and Human Services, “Sequences of proteins ofimmunological interest” (1991)).

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three V_(L) CDRs of an IPN002 antibody; and ii) a humanizedlight chain framework region; and b) a heavy chain region comprising: i)one, two, or three V_(H) CDRs of an IPN002 antibody; and ii) a humanizedheavy chain framework region; where the V_(H) and V_(L) CDRs are asdefined by Chothia (see, e.g., Table 1, above; and Chothia et al., J.Mol. Biol. 196:901-917 (1987)).

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three CDRs selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ IDNO:3; and ii) a humanized light chain framework region; and b) a heavychain region comprising: i) one, two, or three CDRs selected from SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6; and ii) a humanized heavy chainframework region.

In some embodiments, an anti-Tau antibody of the present disclosure(e.g., a subject antibody that specifically binds an epitope in a Taupolypeptide, e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, within amino acids 9 to 18 of Tau (whereamino acids 1-18 of Tau are: MAEPRQEFEVMEDHAGTY; SEQ ID NO:53), withinamino acids 15-44 of Tau, within amino acids 13-24 of Tau, or withinamino acids 15-24 of Tau (where amino acids 15-24 of Tau are: AGTYGLGDRK(SEQ ID NO:51)) comprises: a) a light chain region comprising: i) one,two, or three CDRs selected from SEQ ID NO:7, SEQ ID NO:8, and SEQ IDNO:9; and ii) a humanized light chain framework region; and b) a heavychain region comprising: i) one, two, or three CDRs selected from SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12; and ii) a humanized heavy chainframework region.

In some instances, the antibody comprises: a) a light chain regioncomprising: i) a V_(L) CDR1 comprising an amino acid sequence of SEQ IDNO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising an amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:8; (iii) a V_(L) CDR3 comprising an aminoacid sequence of SEQ ID NO:3 or SEQ ID NO:9; and (iv) a humanized lightchain framework region; and b) a heavy chain region comprising: (i) aV_(H) CDR1 comprising an amino acid sequence of SEQ ID NO:4 or SEQ IDNO:10; (ii) a V_(H) CDR2 comprising an amino acid sequence of SEQ IDNO:5 or SEQ ID NO:11; (iii) a V_(H) CDR3 comprising an amino acidsequence of SEQ ID NO:6 or SEQ ID NO:12; and iv) a humanized heavy chainframework region.

In some embodiments, a subject anti-Tau antibody comprises a heavy chainvariable region comprising one, two, or three of the heavy chain CDRshaving an amino acid sequence selected from one or more of SEQ ID NOs:4,5, and 6; and one, two, three, or four FR regions that are humanized.For example, in some embodiments, a subject antibody comprises a heavychain variable region that comprises, in order from N-terminus toC-terminus: a humanized heavy chain FR1; a CDR1 comprising the aminoacid sequence set forth in SEQ ID NO:4; a humanized heavy chain FR2; aCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; ahumanized heavy chain FR3; a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:6; and a humanized heavy chain FR4.

In some embodiments, a subject anti-Tau antibody comprises a light chainvariable region comprising one, two, or three of the light chain CDRshaving a polypeptide sequence selected from one or more of SEQ ID NOs:1,2, and 3; and one, two, three, or four FR regions that are humanized.For example, in some embodiments, a subject antibody comprises a lightchain variable region that comprises, in order from N-terminus toC-terminus: a humanized light chain FR1; a CDR1 comprising the aminoacid sequence set forth in SEQ ID NO:1; a humanized light chain FR2; aCDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; ahumanized light chain FR3; a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:3; and a humanized light chain FR4.

In some embodiments, a subject anti-Tau antibody comprises a heavy chainvariable region comprising one, two, or three of the heavy chain CDRshaving an amino acid sequence selected from one or more of SEQ IDNOs:10, 11, and 12; and one, two, three, or four FR regions that arehumanized. For example, in some embodiments, a subject antibodycomprises a heavy chain variable region that comprises, in order fromN-terminus to C-terminus: a humanized heavy chain FR1; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO:10; a humanized heavychain FR2; a CDR2 comprising the amino acid sequence set forth in SEQ IDNO:11; a humanized heavy chain FR3; a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:12; and a humanized heavy chain FR4.

In some embodiments, a subject anti-Tau antibody comprises a light chainvariable region comprising one, two, or three of the light chain CDRshaving a polypeptide sequence selected from one or more of SEQ ID NOs:7,8, and 9; and one, two, three, or four FR regions that are humanized.For example, in some embodiments, a subject antibody comprises a lightchain variable region that comprises, in order from N-terminus toC-terminus: a humanized light chain FR1; a CDR1 comprising the aminoacid sequence set forth in SEQ ID NO:7; a humanized light chain FR2; aCDR2 comprising the amino acid sequence set forth in SEQ ID NO:8; ahumanized light chain FR3; a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:9; and a humanized light chain FR4.

V_(H) and V_(L) amino acid sequences of IPN001 are depicted in FIGS. 1Aand 1B. CDRs (as defined by Kabat) are in bold text and underlined.V_(H) and V_(L) amino acid sequences of IPN002 are depicted in FIGS. 2Aand 2B. CDRs (as defined by Kabat) are in bold text and underlined.

SEQ ID NOs:1-12 are as follows:

(SEQ ID NO: 1) RSSQTILHSNGNTYLE; (SEQ ID NO: 2) KVSKRFS; (SEQ ID NO: 3)FQGSLVPWA; (SEQ ID NO: 4) SYGMS; (SEQ ID NO: 5) TISSSGSRTYFPDSVKG;(SEQ ID NO: 6) TWDGAMDY; (SEQ ID NO: 7) KSSQSIVHSNGNTYLE; (SEQ ID NO: 8)KVSNRFS; (SEQ ID NO: 9) FQGSLVPWA; (SEQ ID NO: 10) KYGMS;(SEQ ID NO: 11) TISSSGSRTYYPDSVKG; (SEQ ID NO: 12) SWDGAMDY.

A subject anti-Tau antibody can comprise a light chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 1B and set forth in SEQ ID NO:13.

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 1A and set forth in SEQ ID NO:14.

A subject anti-Tau antibody can comprise a light chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 2B and set forth in SEQ ID NO:15.

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 2A and set forth in SEQ ID NO:16.

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 9 (VH variant 1).

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 10 (VH variant 2).

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 11 (VH variant 3).

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 12 (VH variant 4).

A subject anti-Tau antibody can comprise a light chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 13 (Vk variant 1).

A subject anti-Tau antibody can comprise a light chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 14 (Vk variant 2).

A subject anti-Tau antibody can comprise a light chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 15 (Vk variant 3).

A subject anti-Tau antibody can comprise a light chain variable regioncomprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencedepicted in FIG. 16 (Vk variant 4).

A subject anti-Tau antibody can comprise a heavy chain variable regioncomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the framework(FR) amino acid substitutions, relative to the IPN002 parental antibodyFR amino acid sequences, depicted in Table 2.

TABLE 2 VH Variants IPN002 Amino Acid (Parental VH Vari- VH Vari- VHVari- VH Vari- Position antibody) ant 1 ant 2 ant 3 ant 4 FR1 3 H H H QQ 19 K R R R R FR2 40 T A A A A 42 D G G G G 44 R G G G G FR3 66 Q R R RR 83 S S N N N 85 L S L L L 86 K K R R R 87 S S A A A 93 S S S S A FR4108 S S T T T

For example, a subject anti-Tau antibody can comprise a heavy chainvariable region comprising an H→Q substitution at amino acid position 3in VH FR1 and/or a K→R substitution at amino acid position 19 in VH FR1.

As another example, a subject anti-Tau antibody can comprise a heavychain variable region comprising a T→A substitution at amino acidposition 40 in VH FR2 and/or a D→G substitution at amino acid position42 in VH FR2 and/or an R→G substitution at position 44 in VH FR2.

As another example, a subject anti-Tau antibody can comprise a heavychain variable region comprising a Q→R substitution at amino acidposition 66 in VH FR3 and/or an S→N substitution at amino acid position83 in VH FR3 and/or an L→S substitution at amino acid position 85 in VHFR3 and/or a K→R substitution at amino acid position 86 in FR3 and/or anS-A substitution at amino acid position 87 in VH FR3 and/or an S-Asubstitution at amino acid position 93 in VH FR3.

As another example, a subject anti-Tau antibody can comprise a heavychain variable region comprising an S→T substitution at amino acidposition 108 in VH FR4.

In some cases, a subject isolated anti-Tau antibody can comprise, inorder from N-terminus to C-terminus a VH region comprising:EVX₁LVESGGALVKPGGSLRLSCAASGFSFS (SEQ ID NO:83); VH CDR1 as shown in FIG.2A; WVRQAPGKGLEWVA (SEQ ID NO:84); VH CDR2 as shown in FIG. 2A;RFTISRDNAKNTLYLQMX₂SX₃X₄X₅EDTAMYYCX₆I (SEQ ID NO:85); VH CDR3 as shownin FIG. 2A; WGQGTX₇VTVSS (SEQ ID NO:86), where X₁ is H or Q; X₂ is S orN; X₃ is S or L; X₄ is K or R; X₅ is S or A; X₆ is S or A; and X₇ is Sor T.

A subject anti-Tau antibody can comprise a light chain variable regioncomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the framework (FR) aminoacid substitutions, relative to the IPN002 parental antibody FR aminoacid sequences, depicted in Table 3.

TABLE 3 Vk Variants IPN002 Amino Acid (Parental Vk Vari- Vk Vari- VkVari- Vk Vari- Position antibody) ant 1 ant 2 ant 3 ant 4 FR1 3 L L V VV 7 T S S S S 14 S T T T T 17 D Q Q Q Q 18 Q P P P P FR2 45 K Q Q Q Q 48V V V V I FR3 83 L V V V V 85 T T T V V FR4 104 L V V V V

For example, a subject anti-Tau antibody can comprise a light chainvariable region comprising an L→V substitution at amino acid position 3in VL FR1 and/or a T→S substitution at amino acid position 7 in VL FR1and/or an S→T substitution at amino acid position 14 in VL FR1 and/or aD→Q substitution at amino acid position 17 in VL FR1 and/or a Q→Psubstitution at amino acid position 18 in VL FR1.

As another example, a subject anti-Tau antibody can comprise a lightchain variable region comprising a K→Q substitution at amino acidposition 45 of VL FR2 and/or a V→I substitution at amino acid position48 of VL FR2.

As another example, a subject anti-Tau antibody can comprise a lightchain variable region comprising an L→V substitution at amino acidposition 83 of VL FR3 and/or a T→V substitution at amino acid position85 of VL FR3.

As another example, a subject anti-Tau antibody can comprise a lightchain variable region comprising an L→V substitution at amino acidposition 104 of VL FR4.

In some cases, a subject isolated anti-Tau antibody can comprise, inorder from N-terminus to C-terminus a VL region comprising:DVX₁MTQSPLSLPVTLGQPASISC (SEQ ID NO:54); VL CDR1 as shown in FIG. 2B;WYLQKPGQSPQLLX₂Y (SEQ ID NO:55); VL CDR2 as shown in FIG. 2B;GVPDRFSGSGSGTDFTLKISRVEAEDVGX₃YYC (SEQ ID NO:56); VL CDR3 as shown inFIG. 2B; FGGGTKVEIK (SEQ ID NO:57); where X₁ is L or V; X₂ is V or I;and X₃ is T or V.

In some cases, an anti-Tau antibody of the present disclosure comprises:

a) a VH variant 1 comprising the amino acid sequence depicted in FIG. 9;and a Vk variant 1 comprising the amino acid sequence depicted in FIG.13;

b) a VH variant 1 comprising the amino acid sequence depicted in FIG. 9;and a Vk variant 2 comprising the amino acid sequence depicted in FIG.14;

c) a VH variant 1 comprising the amino acid sequence depicted in FIG. 9;and a Vk variant 3 comprising the amino acid sequence depicted in FIG.15;

d) a VH variant 1 comprising the amino acid sequence depicted in FIG. 9;and a Vk variant 4 comprising the amino acid sequence depicted in FIG.16;

e) a VH variant 2 comprising the amino acid sequence depicted in FIG.10; and a Vk variant 1 comprising the amino acid sequence depicted inFIG. 13;

f) a VH variant 2 comprising the amino acid sequence depicted in FIG.10; and a Vk variant 2 comprising the amino acid sequence depicted inFIG. 14;

g) a VH variant 2 comprising the amino acid sequence depicted in FIG.10; and a Vk variant 3 comprising the amino acid sequence depicted inFIG. 15;

h) a VH variant 2 comprising the amino acid sequence depicted in FIG.10; and a Vk variant 4 comprising the amino acid sequence depicted inFIG. 16;

i) a VH variant 3 comprising the amino acid sequence depicted in FIG.11; and a Vk variant 1 comprising the amino acid sequence depicted inFIG. 13;

j) a VH variant 3 comprising the amino acid sequence depicted in FIG.11; and a Vk variant 2 comprising the amino acid sequence depicted inFIG. 14;

k) a VH variant 3 comprising the amino acid sequence depicted in FIG.11; and a Vk variant 3 comprising the amino acid sequence depicted inFIG. 15;

l) a VH variant 3 comprising the amino acid sequence depicted in FIG.11; and a Vk variant 4 comprising the amino acid sequence depicted inFIG. 16;

m) a VH variant 4 comprising the amino acid sequence depicted in FIG.12; and a Vk variant 1 comprising the amino acid sequence depicted inFIG. 13;

n) a VH variant 4 comprising the amino acid sequence depicted in FIG.12; and a Vk variant 2 comprising the amino acid sequence depicted inFIG. 14;

o) a VH variant 4 comprising the amino acid sequence depicted in FIG.12; and a Vk variant 3 comprising the amino acid sequence depicted inFIG. 15; or

p) a VH variant 4 comprising the amino acid sequence depicted in FIG.12; and a Vk variant 4 comprising the amino acid sequence depicted inFIG. 16.

In some embodiments, a subject antibody comprises anti-Tau heavy chainCDRs and anti-Tau light chain CDRs in a single polypeptide chain, e.g.,in some embodiments, a subject antibody is a scFv. In some embodiments,a subject antibody comprises, in order from N-terminus to C-terminus: afirst amino acid sequence of from about 5 amino acids to about 25 aminoacids in length; a CDR1 comprising the amino acid sequence set forth inSEQ ID NO:1; a second amino acid sequence of from about 5 amino acids toabout 25 amino acids in length; a CDR2 comprising the amino acidsequence set forth in SEQ ID NO:2; a third amino acid sequence of fromabout 5 amino acids to about 25 amino acids in length; a CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO:3; a fourth amino acidsequence of from about 5 amino acids to about 25 amino acids in length;a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4; afifth amino acid sequence of from about 5 amino acids to about 25 aminoacids in length; a CDR2 comprising the amino acid sequence set forth inSEQ ID NO:5; a sixth amino acid sequence of from about 5 amino acids toabout 25 amino acids in length; a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:6; and a seventh amino acid sequence offrom about 5 amino acids to about 25 amino acids in length.

In some embodiments, a subject antibody comprises, in order fromN-terminus to C-terminus: a light chain FR1 region; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO:1; a light chain FR2region; a CDR2 comprising the amino acid sequence set forth in SEQ IDNO:2; a light chain FR3 region; a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:3; optionally a light chain FR4 region;a linker region; optionally a heavy chain FR1 region; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID:4; a heavy chain FR2 region;a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; aheavy chain FR3 region; a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:6; and a heavy chain FR4 region. In some of theseembodiments, one or more of the FR regions is a humanized FR region. Insome of these embodiments, each of the FR regions is a humanized FRregion. The linker region can be from about 5 amino acids to about 50amino acids in length, e.g., from about 5 aa to about 10 aa, from about10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aato about 25 aa, from about 25 aa to about 30 aa, from about 30 aa toabout 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about45 aa, or from about 45 aa to about 50 aa in length.

In some embodiments, a subject antibody comprises, in order fromN-terminus to C-terminus: a heavy chain FR1 region; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID:4; a heavy chain FR2 region;a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; aheavy chain FR3 region; a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:6; optionally a heavy chain FR4 region; a linker;optionally a light chain FR1 region; a CDR1 comprising the amino acidsequence set forth in SEQ ID NO:1; a light chain FR2 region; a CDR2comprising the amino acid sequence set forth in SEQ ID NO:2; a lightchain FR3 region; a CDR3 comprising the amino acid sequence set forth inSEQ ID NO:3; and a light chain FR4 region. In some of these embodiments,one or more of the FR regions is a humanized FR region. In some of theseembodiments, each of the FR regions is a humanized FR region. The linkerregion can be from about 5 amino acids to about 50 amino acids inlength, e.g., from about 5 aa to about 10 aa, from about 10 aa to about15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa,from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, fromabout 35 aa to about 40 aa, from about 40 aa to about 45 aa, or fromabout 45 aa to about 50 aa in length.

In some embodiments, a subject antibody comprises, in order fromN-terminus to C-terminus: a light chain FR1 region; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO:7; a light chain FR2region; a CDR2 comprising the amino acid sequence set forth in SEQ IDNO:8; a light chain FR3 region; a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:9; optionally a light chain FR4 region;a linker region; optionally a heavy chain FR1 region; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID: 10; a heavy chain FR2region; a CDR2 comprising the amino acid sequence set forth in SEQ IDNO:11; a heavy chain FR3 region; a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:12; and a heavy chain FR4 region. Insome of these embodiments, one or more of the FR regions is a humanizedFR region. In some of these embodiments, each of the FR regions is ahumanized FR region. The linker region can be from about 5 amino acidsto about 50 amino acids in length, e.g., from about 5 aa to about 10 aa,from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, fromabout 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aato about 45 aa, or from about 45 aa to about 50 aa in length.

In some embodiments, a subject antibody comprises, in order fromN-terminus to C-terminus: a heavy chain FR1 region; a CDR1 comprisingthe amino acid sequence set forth in SEQ ID: 10; a heavy chain FR2region; a CDR2 comprising the amino acid sequence set forth in SEQ IDNO:11; a heavy chain FR3 region; a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:12; optionally a heavy chain FR4 region;a linker; optionally a light chain FR1 region; a CDR1 comprising theamino acid sequence set forth in SEQ ID NO:7; a light chain FR2 region;a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8; alight chain FR3 region; a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:9; and a light chain FR4 region. In some of theseembodiments, one or more of the FR regions is a humanized FR region. Insome of these embodiments, each of the FR regions is a humanized FRregion. The linker region can be from about 5 amino acids to about 50amino acids in length, e.g., from about 5 aa to about 10 aa, from about10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aato about 25 aa, from about 25 aa to about 30 aa, from about 30 aa toabout 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about45 aa, or from about 45 aa to about 50 aa in length.

Linkers suitable for use a subject antibody include “flexible linkers”.If present, the linker molecules are generally of sufficient length topermit some flexible movement between linked regions. The linkermolecules are generally about 6-50 atoms long. The linker molecules mayalso be, for example, aryl acetylene, ethylene glycol oligomerscontaining 2-10 monomer units, diamines, diacids, amino acids, orcombinations thereof. Other linker molecules which can bind topolypeptides may be used in light of this disclosure.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO:58) and GGGS_(n) (SEQ ID NO:59), where n is an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers known in the art. Glycine and glycine-serine polymersare of interest since both of these amino acids are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Glycine polymers are of particular interest since glycineaccesses significantly more phi-psi space than even alanine, and is muchless restricted than residues with longer side chains (see Scheraga,Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkersinclude, but are not limited GGSG (SEQ ID NO:60), GGSGG (SEQ ID NO:61),GSGSG (SEQ ID NO:62), GSGGG (SEQ ID NO:63), GGGSG (SEQ ID NO:64), GSSSG(SEQ ID NO:65), and the like. The ordinarily skilled artisan willrecognize that design of a peptide conjugated to any elements describedabove can include linkers that are all or partially flexible, such thatthe linker can include a flexible linker as well as one or more portionsthat confer less flexible structure.

In some cases, a subject isolated antibody is an antibody fragment, anFv, scFv, Fab, F(ab′)2, or Fab′. Thus, the present disclosure providesan isolated antibody, wherein the antibody is a Fv, scFv, Fab, F(ab′)2,or Fab′, and wherein the antibody competes for binding to an epitope inan N-terminal region of a Tau polypeptide with an antibody thatcomprises: a) a light chain region comprising: i) a V_(L) CDR1comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) aV_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8; and (iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H)CDR1 comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10;(ii) a V_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 orSEQ ID NO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequenceof SEQ ID NO:6 or SEQ ID NO:12. In some of these embodiments, theisolated antibody comprises one, two, three, or four humanized VLframework regions, as described above. In some of these embodiments, theisolated antibody comprises one, two, three, or four humanized VHframework regions, as described above.

In some embodiments, an anti-Tau antibody of the present disclosure is ascFv antibody. In some embodiments, an anti-Tau antibody of the presentdisclosure comprises scFv multimers. For example, in some embodiments, asubject antibody is an scFv dimer (e.g., comprises two tandem scFv(scFv₂)), an scFv trimer (e.g., comprises three tandem scFv (scFv₃)), anscFv tetramer (e.g., comprises four tandem scFv (scFv₄)), or is amultimer of more than four scFv (e.g., in tandem). The scFv monomers canbe linked in tandem via linkers of from about 2 amino acids to about 10amino acids (aa) in length, e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8aa, 9 aa, or 10 aa in length. Suitable linkers include, e.g., (Gly)_(x),where x is an integer from 2 to 10. Other suitable linkers are thosediscussed above. In some embodiments, each of the scFv monomers in asubject scFV multimer is humanized, as described above.

In some cases, a subject antibody comprises a constant region of animmunoglobulin (e.g., an Fc region). The Fc region, if present, can be ahuman Fc region. If constant regions are present, the antibody cancontain both light chain and heavy chain constant regions. Suitableheavy chain constant region include CH1, hinge, CH2, CH3, and CH4regions. The antibodies described herein include antibodies having alltypes of constant regions, including IgM, IgG, IgD, IgA and IgE, and anyisotype, including IgG1, IgG2, IgG3 and IgG4. An example of a suitableheavy chain Fc region is a human isotype IgG1 Fc. In some cases, theheavy chain region is of the isotype IgG4. In some of these embodiments,the hinge region comprises an S241P substitution. See, e.g., Angal etal. (1993) Mol. Immunol. 30:105. Light chain constant regions can belambda or kappa. A subject antibody (e.g., a subject humanized antibody)can comprise sequences from more than one class or isotype. Antibodiescan be expressed as tetramers containing two light and two heavy chains,as separate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, oras single chain antibodies in which heavy and light chain variabledomains are linked through a spacer.

In some embodiments, the present disclosure provides an isolatedantibody, wherein the isolated antibody comprises a human light chainconstant region and a human heavy chain constant region, and wherein theisolated antibody competes for binding to an epitope in an N-terminalregion of a Tau polypeptide with an antibody that comprises: a) a lightchain region comprising: i) a V_(L) CDR1 comprising an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:8; and (iii) a V_(L)CDR3 comprising an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:9;and b) a heavy chain region comprising: (i) a V_(H) CDR1 comprising anamino acid sequence of SEQ ID NO:4 or SEQ ID NO:10; (ii) a V_(H) CDR2comprising an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:11; and(iii) a V_(H) CDR3 comprising an amino acid sequence of SEQ ID NO:6 orSEQ ID NO:12. In some of these embodiments, the isolated antibodycomprises one, two, three, or four humanized VL framework regions, asdescribed above. In some of these embodiments, the isolated antibodycomprises one, two, three, or four humanized VH framework regions, asdescribed above.

A subject antibody can comprise a free thiol (—SH) group at the carboxylterminus, where the free thiol group can be used to attach the antibodyto a second polypeptide (e.g., another antibody, including a subjectantibody), a scaffold, a carrier, etc.

In some embodiments, a subject antibody comprises one or morenon-naturally occurring amino acids. In some embodiments, thenon-naturally encoded amino acid comprises a carbonyl group, an acetylgroup, an aminooxy group, a hydrazine group, a hydrazide group, asemicarbazide group, an azide group, or an alkyne group. See, e.g., U.S.Pat. No. 7,632,924 for suitable non-naturally occurring amino acids.Inclusion of a non-naturally occurring amino acid can provide forlinkage to a polymer, a second polypeptide, a scaffold, etc. Forexample, a subject antibody linked to a water-soluble polymer can bemade by reacting a water-soluble polymer (e.g., PEG) that comprises acarbonyl group to the antibody, where the antibody comprises anon-naturally encoded amino acid that comprises an aminooxy, hydrazine,hydrazide or semicarbazide group. As another example, a subject antibodylinked to a water-soluble polymer can be made by reacting a subjectantibody that comprises an alkyne-containing amino acid with awater-soluble polymer (e.g., PEG) that comprises an azide moiety; insome embodiments, the azide or alkyne group is linked to the PEGmolecule through an amide linkage. A “non-naturally encoded amino acid”refers to an amino acid that is not one of the 20 common amino acids orpyrrolysine or selenocysteine. Other terms that may be used synonymouslywith the term “non-naturally encoded amino acid” are “non-natural aminoacid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” andvariously hyphenated and non-hyphenated versions thereof. The term“non-naturally encoded amino acid” also includes, but is not limited to,amino acids that occur by modification (e.g. post-translationalmodifications) of a naturally encoded amino acid (including but notlimited to, the 20 common amino acids or pyrrolysine and selenocysteine)but are not themselves naturally incorporated into a growing polypeptidechain by the translation complex. Examples of suchnon-naturally-occurring amino acids include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine.

In some embodiments, a subject antibody is linked (e.g., covalentlylinked) to a polymer (e.g., a polymer other than a polypeptide).Suitable polymers include, e.g., biocompatible polymers, andwater-soluble biocompatible polymers. Suitable polymers includesynthetic polymers and naturally-occurring polymers. Suitable polymersinclude, e.g., substituted or unsubstituted straight or branched chainpolyalkylene, polyalkenylene or polyoxyalkylene polymers or branched orunbranched polysaccharides, e.g. a homo- or hetero-polysaccharide.Suitable polymers include, e.g., ethylene vinyl alcohol copolymer(commonly known by the generic name EVOH or by the trade name EVAL);polybutylmethacrylate; poly(hydroxyvalerate); poly(L-lactic acid);polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate);poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester;polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolicacid-co-trimethylene carbonate); polyphosphoester; polyphosphoesterurethane; poly(amino acids); cyanoacrylates; poly(trimethylenecarbonate); poly(iminocarbonate); copoly(ether-esters) (e.g.,poly(ethylene oxide)-poly(lactic acid) (PEO/PLA) co-polymers);polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin,fibrinogen, cellulose, starch, collagen and hyaluronic acid;polyurethanes; silicones; polyesters; polyolefins; polyisobutylene andethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinylhalide polymers and copolymers, such as polyvinyl chloride; polyvinylethers, such as polyvinyl methyl ether; polyvinylidene halides, such aspolyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile;polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinylesters, such as polyvinyl acetate; copolymers of vinyl monomers witheach other and olefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetatecopolymers; polyamides, such as Nylon 66 and polycaprolactam; alkydresins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxyresins; polyurethanes; rayon; rayon-triacetate; cellulose; celluloseacetate; cellulose butyrate; cellulose acetate butyrate; cellophane;cellulose nitrate; cellulose propionate; cellulose ethers; amorphousTeflon; poly(ethylene glycol); and carboxymethyl cellulose.

Suitable synthetic polymers include unsubstituted and substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol), and derivatives thereof, e.g., substitutedpoly(ethyleneglycol) such as methoxypoly(ethyleneglycol), andderivatives thereof. Suitable naturally-occurring polymers include,e.g., albumin, amylose, dextran, glycogen, and derivatives thereof.

Suitable polymers can have an average molecular weight in a range offrom 500 Da to 50000 Da, e.g., from 5000 Da to 40000 Da, or from 25000to 40000 Da. For example, in some embodiments, where a subject antibodycomprises a poly(ethylene glycol) (PEG) or methoxypoly(ethyleneglycol)polymer, the PEG or methoxypoly(ethyleneglycol) polymer can have amolecular weight in a range of from about 0.5 kiloDaltons (kDa) to 1kDa, from about 1 kDa to 5 kDa, from 5 kDa to 10 kDa, from 10 kDa to 25kDa, from 25 kDa to 40 kDa, or from 40 kDa to 60 kDa.

As noted above, in some embodiments, a subject antibody is covalentlylinked to a PEG polymer. In some embodiments, a subject scFv multimer iscovalently linked to a PEG polymer. See, e.g., Albrecht et al. (2006) J.Immunol. Methods 310:100. Methods and reagents suitable for PEGylationof a protein are well known in the art and may be found in, e.g., U.S.Pat. No. 5,849,860. PEG suitable for conjugation to a protein isgenerally soluble in water at room temperature, and has the generalformula R(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective groupsuch as an alkyl or an alkanol group, and where n is an integer from 1to 1000. Where R is a protective group, it generally has from 1 to 8carbons.

The PEG conjugated to the subject antibody can be linear. The PEGconjugated to the subject protein may also be branched. Branched PEGderivatives such as those described in U.S. Pat. No. 5,643,575,“star-PEG's” and multi-armed PEG's such as those described in ShearwaterPolymers, Inc. catalog “Polyethylene Glycol Derivatives 1997-1998.” StarPEGs are described in the art including, e.g., in U.S. Pat. No.6,046,305.

A subject antibody can be glycosylated, e.g., a subject antibody cancomprise a covalently linked carbohydrate or polysaccharide moiety.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to an antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of an antibody.

A subject antibody will in some embodiments comprise a “radiopaque”label, e.g. a label that can be easily visualized using for examplex-rays. Radiopaque materials are well known to those of skill in theart. The most common radiopaque materials include iodide, bromide orbarium salts. Other radiopaque materials are also known and include, butare not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No.5,939,045), radiopaque multiurethanes (see U.S. Pat. No. 5,346,981),organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radiopaque barium multimer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

A subject antibody can be covalently linked to a second moiety (e.g., alipid, a polypeptide other than a subject antibody, a synthetic polymer,a carbohydrate, and the like) using for example, glutaraldehyde, ahomobifunctional cross-linker, or a heterobifunctional cross-linker.Glutaraldehyde cross-links polypeptides via their amino moieties.Homobifunctional cross-linkers (e.g., a homobifunctional imidoester, ahomobifunctional N-hydroxysuccinimidyl (NHS) ester, or ahomobifunctional sulfhydryl reactive cross-linker) contain two or moreidentical reactive moieties and can be used in a one-step reactionprocedure in which the cross-linker is added to a solution containing amixture of the polypeptides to be linked. Homobifunctional NHS ester andimido esters cross-link amine containing polypeptides. In a mildalkaline pH, imido esters react only with primary amines to formimidoamides, and overall charge of the cross-linked polypeptides is notaffected. Homobifunctional sulfhydryl reactive cross-linkers includesbismaleimidhexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and1,4-di-(3′,2′-pyridyldithio) propinoamido butane (DPDPB).

Heterobifunctional cross-linkers have two or more different reactivemoieties (e.g., amine reactive moiety and a sulfhydryl-reactive moiety)and are cross-linked with one of the polypeptides via the amine orsulfhydryl reactive moiety, then reacted with the other polypeptide viathe non-reacted moiety. Multiple heterobifunctional haloacetylcross-linkers are available, as are pyridyl disulfide cross-linkers.Carbodiimides are a classic example of heterobifunctional cross-linkingreagents for coupling carboxyls to amines, which results in an amidebond.

A subject antibody can be immobilized on a solid support. Suitablesupports are well known in the art and comprise, inter alia,commercially available column materials, polystyrene beads, latex beads,magnetic beads, colloid metal particles, glass and/or silicon chips andsurfaces, nitrocellulose strips, nylon membranes, sheets, duracytes,wells of reaction trays (e.g., multi-well plates), plastic tubes, etc. Asolid support can comprise any of a variety of substances, including,e.g., glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amylose, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Suitablemethods for immobilizing a subject antibody onto a solid support arewell known and include, but are not limited to ionic, hydrophobic,covalent interactions and the like. Solid supports can be soluble orinsoluble, e.g., in aqueous solution. In some embodiments, a suitablesolid support is generally insoluble in an aqueous solution.

A subject antibody will in some embodiments comprise a detectable label.Suitable detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable include, but are not limited to,magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, a green fluorescent protein, a redfluorescent protein, a yellow fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and others commonlyused in an enzyme-linked immunosorbent assay (ELISA)), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.polystyrene, polypropylene, latex, etc.) beads.

In some embodiments, a subject antibody comprises a contrast agent or aradioisotope, where the contrast agent or radioisotope is one that issuitable for use in imaging, e.g., imaging procedures carried out onhumans. Non-limiting examples of labels include radioisotope such as¹²³¹I (iodine), ¹⁸F (fluorine), ⁹⁹Tc (technetium), ¹¹¹In (indium), and⁶⁷Ga (gallium), and contrast agent such as gadolinium (Gd), dysprosium,and iron. Radioactive Gd isotopes (¹⁵³Gd) also are available andsuitable for imaging procedures in non-human mammals. A subject antibodycan be labeled using standard techniques. For example, a subjectantibody can be iodinated using chloramine T or1,3,4,6-tetrachloro-3α,6α-diphenylglycouril. For fluorination, fluorineis added to a subject antibody during the synthesis by a fluoride iondisplacement reaction. See, Muller-Gartner, H., TIB Tech., 16:122-130(1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244(1999) for a review of synthesis of proteins with such radioisotopes. Asubject antibody can also be labeled with a contrast agent throughstandard techniques. For example, a subject antibody can be labeled withGd by conjugating low molecular Gd chelates such as Gd diethylenetriamine pentaacetic acid (GdDTPA) or Gdtetraazacyclododecanetetraacetic (GdDOTA) to the antibody. See, Caravanet al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn.Reson. Imaging, 3:11-16 (1985). A subject antibody can be labeled withGd by, for example, conjugating polylysine-Gd chelates to the antibody.See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998).Alternatively, a subject antibody can be labeled with Gd by incubatingparamagnetic polymerized liposomes that include Gd chelator lipid withavidin and biotinylated antibody. See, for example, Sipkins et al.,Nature Med., 4:623-626 (1998).

Suitable fluorescent proteins that can be linked to a subject antibodyinclude, but are not limited to, a green fluorescent protein fromAequoria victoria or a mutant or derivative thereof e.g., as describedin U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750;5,968,738; 5,958,713; 5,919,445; 5,874,304; e.g., Enhanced GFP, manysuch GFP which are available commercially, e.g., from Clontech, Inc.; ared fluorescent protein; a yellow fluorescent protein; any of a varietyof fluorescent and colored proteins from Anthozoan species, as describedin, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and thelike.

A subject antibody will in some embodiments be linked to (e.g.,covalently or non-covalently linked) a fusion partner, e.g., a ligand;an epitope tag; a peptide; a protein other than an antibody; and thelike. Suitable fusion partners include peptides and polypeptides thatconfer enhanced stability in vivo (e.g., enhanced serum half-life);provide ease of purification, e.g., (His)_(n), e.g., 6His, and the like;provide for secretion of the fusion protein from a cell; provide anepitope tag, e.g., GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ IDNO:71), FLAG (e.g., DYKDDDDK; SEQ ID NO:69), c-myc (e.g., EQKLISEEDL;SEQ ID NO:68), and the like; provide a detectable signal, e.g., anenzyme that generates a detectable product (e.g., β-galactosidase,luciferase), or a protein that is itself detectable, e.g., a greenfluorescent protein, a red fluorescent protein, a yellow fluorescentprotein, etc.; provides for multimerization, e.g., a multimerizationdomain such as an Fc portion of an immunoglobulin; and the like.

The fusion may also include an affinity domain, including peptidesequences that can interact with a binding partner, e.g., such as oneimmobilized on a solid support, useful for identification orpurification. Consecutive single amino acids, such as histidine, whenfused to a protein, can be used for one-step purification of the fusionprotein by high affinity binding to a resin column, such as nickelsepharose. Exemplary affinity domains include His5 (HHHHH) (SEQ IDNO:66), His×6 (HHHHHH) (SEQ ID NO:67), C-myc (EQKLISEEDL) (SEQ IDNO:68), Flag (DYKDDDDK) (SEQ ID NO:69), StrepTag (WSHPQFEK) (SEQ IDNO:70), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:71),glutathinone-S-transferase (GST), thioredoxin, cellulose binding domain,RYIRS (SEQ ID NO:72), Phe-His-His-Thr (SEQ ID NO:73), chitin bindingdomain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,WEAAAREACCRECCARA (SEQ ID NO:74), metal binding domains, e.g., zincbinding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin,inteins, biotin, streptavidin, MyoD, leucine zipper sequences, andmaltose binding protein.

A subject antibody will in some embodiments be fused to a polypeptidethat binds to an endogenous blood brain barrier (BBB) receptor. Linkinga subject antibody to a polypeptide that binds to an endogenous BBBreceptor facilitates crossing the BBB, e.g., in a subject treatmentmethod (see below) involving administration of a subject antibody to anindividual in need thereof. Suitable polypeptides that bind to anendogenous BBB receptor include antibodies, e.g., monoclonal antibodies,or antigen-binding fragments thereof, that specifically bind to anendogenous BBB receptor. Suitable endogenous BBB receptors include, butare not limited to, an insulin receptor, a transferrin receptor, aleptin receptor, a lipoprotein receptor, and an insulin-like growthfactor receptor. See, e.g., U.S. Patent Publication No. 2009/0156498.

As an example, a subject anti-Tau antibody can be a bi-specific antibodycomprising a first antigen-binding portion that specifically binds anepitope in a Tau polypeptide (e.g., a linear epitope within anamino-terminal (N-terminal) portion of Tau, e.g., within amino acids1-25 of Tau, within amino acids 1-18 of Tau, or within amino acids 9 to18 of Tau); and a second antigen-binding portion that binds anendogenous BBB receptor. For example, in some instances, a subjectanti-Tau antibody is a bi-specific antibody comprising a firstantigen-binding portion that specifically binds an epitope in a Taupolypeptide (e.g., a linear epitope within an amino-terminal(N-terminal) portion of Tau, e.g., within amino acids 1-25 of Tau,within amino acids 1-18 of Tau, or within amino acids 9 to 18 of Tau);and a second antigen-binding portion that binds a transferrin receptor.

For example, an anti-Tau antibody of the present disclosure can be fusedto a peptide that facilitates crossing the BBB, the peptide having alength of from about 15 amino acids to about 25 amino acids, andcomprising an amino acid sequence having at least about 85% amino acidsequence identity to one of the following peptides: Angiopep-1(TFFYGGCRGKRNNFKTEEY; SEQ ID NO:75); Angiopep-2 (TFFYGGSRGKRNNFKTEEY;SEQ ID NO:76); cys-Angiopep-2 (CTFFYGGSRGKRNNFKTEEY; SEQ ID NO:77);Angiopep-2-cys (TFFYGGSRGKRNNFKTEEYC; SEQ ID NO:78); and an aprotininfragment (TFVYGGCRAKRNNFKS; SEQ ID NO:79). See, e.g., U.S. PatentPublication Nos. 2011/0288011; and 2009/0016959. A peptide thatfacilitates crossing the BBB can be fused to the N-terminus of ananti-Tau light chain region, to the C-terminus of an anti-Tau lightchain region, to the N-terminus of an anti-Tau heavy chain region, tothe C-terminus of an anti-Tau heavy chain region, to the N-terminus of asubject anti-Tau single-chain antibody, to the C-terminus of a subjectanti-Tau single-chain antibody, etc.

In some embodiments, a subject antibody comprises a polyaminemodification. Polyamine modification of a subject antibody enhancespermeability of the modified antibody at the BBB. A subject antibody canbe modified with polyamines that are either naturally occurring orsynthetic. See, for example, U.S. Pat. No. 5,670,477. Useful naturallyoccurring polyamines include putrescine, spermidine, spermine,1,3-diaminopropane, norspermidine, syn-homospermidine, thermine,thermospermine, caldopentamine, homocaldopentamine, and canavalmine.Putrescine, spermidine and spermine are particularly useful. Syntheticpolyamines are composed of the empirical formula C_(X)H_(Y)N_(Z), can becyclic or acyclic, branched or unbranched, hydrocarbon chains of 3-12carbon atoms that further include 1-6 NR or N(R)₂ moieties, wherein R isH, (C₁-C₄) alkyl, phenyl, or benzyl. Polyamines can be linked to anantibody using any standard crosslinking method.

In some embodiments, a subject antibody is modified to include acarbohydrate moiety, where the carbohydrate moiety can be covalentlylinked to the antibody. In some embodiments, a subject antibody ismodified to include a lipid moiety, where the lipid moiety can becovalently linked to the antibody. Suitable lipid moieties include,e.g., an N-fatty acyl group such as N-lauroyl, N-oleoyl, etc.; a fattyamine such as dodecyl amine, oleoyl amine, etc.; a C3-C16 long-chainaliphatic lipid; and the like. See, e.g., U.S. Pat. No. 6,638,513). Insome embodiments, a subject antibody is incorporated into a liposome.

Methods of Producing a Subject Antibody

A subject antibody can be produced by any known method, e.g.,conventional synthetic methods for protein synthesis; recombinant DNAmethods; etc.

Where a subject antibody is a single chain polypeptide, it can besynthesized using standard chemical peptide synthesis techniques. Wherea polypeptide is chemically synthesized, the synthesis may proceed vialiquid-phase or solid-phase. Solid phase polypeptide synthesis (SPPS),in which the C-terminal amino acid of the sequence is attached to aninsoluble support followed by sequential addition of the remaining aminoacids in the sequence, is an example of a suitable method for thechemical synthesis of a subject antibody. Various forms of SPPS, such asFmoc and Boc, are available for synthesizing a subject antibody.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156(1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. MedChem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8.Briefly, small insoluble, porous beads are treated with functional unitson which peptide chains are built. After repeated cycling ofcoupling/deprotection, the free N-terminal amine of a solid-phaseattached is coupled to a single N-protected amino acid unit. This unitis then deprotected, revealing a new N-terminal amine to which a furtheramino acid may be attached. The peptide remains immobilized on thesolid-phase and undergoes a filtration process before being cleaved off.

Standard recombinant methods can be used for production of a subjectantibody. For example, nucleic acids encoding light and heavy chainvariable regions, optionally linked to constant regions, are insertedinto expression vectors. The light and heavy chains can be cloned in thesame or different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. The expression control sequences can be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells (e.g., COS or CHO cells). Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the antibodies.

Because of the degeneracy of the code, a variety of nucleic acidsequences can encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de novo solid-phaseDNA synthesis or by polymerase chain reaction (PCR) mutagenesis of anearlier prepared variant of the desired polynucleotide.Oligonucleotide-mediated mutagenesis is an example of a suitable methodfor preparing substitution, deletion and insertion variants of targetpolypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, thetarget polypeptide DNA is altered by hybridizing an oligonucleotideencoding the desired mutation to a single-stranded DNA template. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that incorporates theoligonucleotide primer, and encodes the selected alteration in thetarget polypeptide DNA.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markers(e.g., ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance) to permitdetection of those cells transformed with the desired DNA sequences.

Escherichia coli is an example of a prokaryotic host cell that can beused for cloning a subject antibody-encoding polynucleotide. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilis, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, with suitable vectors having expression controlsequences (e.g., promoters), an origin of replication, terminationsequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase,isocytochrome C, and enzymes responsible for maltose and galactoseutilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cellsgrown in in vitro cell culture) can also be used to express and producean anti-tau antibody of the present disclosure (e.g., polynucleotidesencoding a subject anti-Tau antibody). See Winnacker, From Genes toClones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cellsinclude CHO cell lines, various Cos cell lines, HeLa cells, myeloma celllines, and transformed B-cells or hybridomas. Expression vectors forthese cells can include expression control sequences, such as an originof replication, a promoter, and an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Examples of suitable expressioncontrol sequences are promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Coet al., J. Immunol. 148:1149 (1992).

Once synthesized (either chemically or recombinantly), the wholeantibodies, their dimers, individual light and heavy chains, or otherforms of a subject antibody (e.g., scFv, etc.) can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, high performanceliquid chromatography (HPLC) purification, gel electrophoresis, and thelike (see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,(1982)). A subject antibody can be substantially pure, e.g., at leastabout 80% to 85% pure, at least about 85% to 90% pure, at least about90% to 95% pure, or 98% to 99%, or more, pure, e.g., free fromcontaminants such as cell debris, macromolecules other than a subjectantibody, etc.

Compositions

The present disclosure provides a composition comprising a subjectantibody. A subject antibody composition can comprise, in addition to asubject antibody, one or more of: a salt, e.g., NaCl, MgCl₂, KCl, MgSO₄,etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

Nucleic Acids, Expression Vectors, and Host Cells

The present disclosure provides nucleic acids comprising nucleotidesequences encoding a subject anti-Tau antibody.

A nucleotide sequence encoding subject anti-Tau antibody can comprise anucleotide sequence encoding a light chain variable region and having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% nucleotide sequence identity to the nucleotide sequencedepicted in FIG. 1B and set forth in SEQ ID NO:17.

A nucleotide sequence encoding subject anti-Tau antibody can comprise anucleotide sequence encoding a heavy chain variable region and having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% nucleotide sequence identity to the nucleotide sequencedepicted in FIG. 1A and set forth in SEQ ID NO:18.

A nucleotide sequence encoding subject anti-Tau antibody can comprise anucleotide sequence encoding a light chain variable region and having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% nucleotide sequence identity to the nucleotide sequencedepicted in FIG. 2B and set forth in SEQ ID NO:19.

A nucleotide sequence encoding subject anti-Tau antibody can comprise anucleotide sequence encoding a heavy chain variable region and having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% nucleotide sequence identity to the nucleotide sequencedepicted in FIG. 2A and set forth in SEQ ID NO:20.

A nucleotide sequence encoding a subject antibody can be operably linkedto one or more regulatory elements, such as a promoter and enhancer,that allow expression of the nucleotide sequence in the intended targetcells (e.g., a cell that is genetically modified to synthesize theencoded antibody).

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC 1 promoter, a TRP 1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637),apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1):86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirBpromoter (Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like(see, e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvieet al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992)Biotechnol. 10:888-892); a sigma70 promoter, e.g., a consensus sigma70promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, andAX798183); a stationary phase promoter, e.g., a dps promoter, an spypromoter, and the like; a promoter derived from the pathogenicity islandSPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Ramaet al. (2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the LacI repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a subject antibody can be present in anexpression vector and/or a cloning vector. Where a subject antibodycomprises two separate polypeptides, nucleotide sequences encoding thetwo polypeptides can be cloned in the same or separate vectors. Anexpression vector can include a selectable marker, an origin ofreplication, and other features that provide for replication and/ormaintenance of the vector.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., InvestOpthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., HGene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see,e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al.,Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989)63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte etal., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; humanimmunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector(e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derivedfrom retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, human immunodeficiency virus, myeloproliferativesarcoma virus, and mammary tumor virus); and the like.

As noted above, a subject nucleic acid comprises a nucleotide sequenceencoding a subject antibody. A subject nucleic acid can comprise anucleotide sequence encoding heavy- and light-chain CDRs of IPN001. Insome embodiments, a subject nucleic acid comprises a nucleotide sequenceencoding heavy- and light-chain CDRs of IPN002, where the CDR-encodingsequences are interspersed with FR-encoding nucleotide sequences. Insome embodiments, the FR-encoding nucleotide sequences are humanFR-encoding nucleotide sequences.

Host Cells

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a subjectnucleic acid. In some embodiments, a subject isolated geneticallymodified host cell can produce a subject antibody.

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like. In some cases, the cells are HEKcells. In some cases, the cells are CHO cells, e.g., CHO-K1 cells (ATCCNo. CCL-61), CHO-M cells, CHO-DG44 cells (ATCC No. PTA-3356), and thelike.

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,and the like. See, e.g., Carrier et al. (1992) J. Immunol.148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemore et al. (1995)Science 270:299-302. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, and the like. Insome embodiments, the host cell is Escherichia coli.

Pharmaceutical Formulations

The present disclosure provides compositions, including pharmaceuticalcompositions, comprising a subject antibody. In general, a formulationcomprises an effective amount of a subject antibody. An “effectiveamount” means a dosage sufficient to produce a desired result, e.g.,reduction in an adverse symptom associated with a tauopathy,amelioration of a symptom of a tauopathy, slowing progression of atauopathy, etc. Generally, the desired result is at least a reduction ina symptom of a tauopathy, as compared to a control. A subject antibodycan be delivered in such a manner as to avoid the blood-brain barrier,as described in more detail below. A subject antibody can be formulatedand/or modified to enable the antibody to cross the blood-brain barrier.

The present disclosure provides a pharmaceutical formulation comprising:a) an antibody that specifically binds an epitope within an N-terminalportion of Tau, wherein the antibody comprises: (i) a V_(L) CDR1comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) aV_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8; (iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:9; (iv) a V_(H) CDR1 comprising an amino acid sequenceof SEQ ID NO:4 or SEQ ID NO:10; (v) a V_(H) CDR2 comprising an aminoacid sequence of SEQ ID NO:5 or SEQ ID NO:11; and (vi) a V_(H) CDR3comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:12; and b)a pharmaceutically acceptable excipient suitable for administration to ahuman, wherein the formulation is free of endotoxins.

The present disclosure provides a pharmaceutical formulation comprising:a) an isolated humanized monoclonal antibody that specifically binds anepitope within amino acids 15-24 of a Tau polypeptide; and b) apharmaceutically acceptable excipient, where in some embodiments thepharmaceutically acceptable excipient is suitable for administration toa human.

The present disclosure provides a pharmaceutical formulation comprising:A) an isolated antibody comprising a humanized light chain frameworkregion; and a humanized heavy chain framework region, wherein theisolated antibody competes for binding to an epitope in an N-terminalregion of a Tau polypeptide with an antibody that comprises: a) a lightchain region comprising: i) a V_(L) CDR1 comprising an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2 comprising anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:8; and (iii) a V_(L)CDR3 comprising an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:9;and b) a heavy chain region comprising: (i) a V_(H) CDR1 comprising anamino acid sequence of SEQ ID NO:4 or SEQ ID NO:10; (ii) a V_(H) CDR2comprising an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:11; and(iii) a V_(H) CDR3 comprising an amino acid sequence of SEQ ID NO:6 orSEQ ID NO:12; and B) a pharmaceutically acceptable excipient, where insome embodiments the pharmaceutically acceptable excipient is suitablefor administration to a human.

The present disclosure provides a pharmaceutical formulation comprising:A) an isolated antibody, wherein the antibody is a Fv, scFv, Fab,F(ab′)2, or Fab′, and wherein the antibody competes for binding to anepitope in an N-terminal region of a Tau polypeptide with an antibodythat comprises: a) a light chain region comprising: i) a V_(L) CDR1comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) aV_(L) CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8; and (iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H)CDR1 comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10;(ii) a V_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 orSEQ ID NO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequenceof SEQ ID NO:6 or SEQ ID NO:12; and B) a pharmaceutically acceptableexcipient, where in some embodiments the pharmaceutically acceptableexcipient is suitable for administration to a human.

The present disclosure provides a pharmaceutical formulation comprising:A) an isolated antibody, wherein the isolated antibody comprises a humanlight chain constant region and a human heavy chain constant region, andwherein the isolated antibody competes for binding to an epitope in anN-terminal region of a Tau polypeptide with an antibody that comprises:a) a light chain region comprising: i) a V_(L) CDR1 comprising an aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:7; (ii) a V_(L) CDR2comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8; and(iii) a V_(L) CDR3 comprising an amino acid sequence of SEQ ID NO:3 orSEQ ID NO:9; and b) a heavy chain region comprising: (i) a V_(H) CDR1comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10; (ii) aV_(H) CDR2 comprising an amino acid sequence of SEQ ID NO:5 or SEQ IDNO:11; and (iii) a V_(H) CDR3 comprising an amino acid sequence of SEQID NO:6 or SEQ ID NO:12; and B) a pharmaceutically acceptable excipient,where in some embodiments the pharmaceutically acceptable excipient issuitable for administration to a human.

Formulations

In the subject methods, a subject antibody can be administered to thehost using any convenient means capable of resulting in the desiredtherapeutic effect or diagnostic effect. Thus, the agent can beincorporated into a variety of formulations for therapeuticadministration. More particularly, a subject antibody can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, a subject antibody can be administeredin the form of their pharmaceutically acceptable salts, or they may alsobe used alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, a subject antibody can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

A subject antibody can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

Pharmaceutical compositions comprising a subject antibody are preparedby mixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, stabilizers,surfactants, buffers and/or tonicity agents. Acceptable carriers,excipients and/or stabilizers are nontoxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,glutathione, cysteine, methionine and citric acid; preservatives (suchas ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylor propyl parabens, benzalkonium chloride, or combinations thereof);amino acids such as arginine, glycine, ornithine, lysine, histidine,glutamic acid, aspartic acid, isoleucine, leucine, alanine,phenylalanine, tyrosine, tryptophan, methionine, serine, proline andcombinations thereof; monosaccharides, disaccharides and othercarbohydrates; low molecular weight (less than about 10 residues)polypeptides; proteins, such as gelatin or serum albumin; chelatingagents such as EDTA; sugars such as trehalose, sucrose, lactose,glucose, mannose, maltose, galactose, fructose, sorbose, raffinose,glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid;and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, orpolyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, wherein thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization); howeversolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration; see alsoChen (1992) Drug Dev Ind Pharm 18, 1311-54.

Exemplary antibody concentrations in a subject pharmaceuticalcomposition may range from about 1 mg/mL to about 200 mg/ml or fromabout 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200mg/mL.

An aqueous formulation of the antibody may be prepared in a pH-bufferedsolution, e.g., at pH ranging from about 4.0 to about 7.0, or from about5.0 to about 6.0, or alternatively about 5.5. Examples of buffers thatare suitable for a pH within this range include phosphate-, histidine-,citrate-, succinate-, acetate-buffers and other organic acid buffers.The buffer concentration can be from about 1 mM to about 100 mM, or fromabout 5 mM to about 50 mM, depending, e.g., on the buffer and thedesired tonicity of the formulation.

A tonicity agent may be included in the antibody formulation to modulatethe tonicity of the formulation. Exemplary tonicity agents includesodium chloride, potassium chloride, glycerin and any component from thegroup of amino acids, sugars as well as combinations thereof. In someembodiments, the aqueous formulation is isotonic, although hypertonic orhypotonic solutions may be suitable. The term “isotonic” denotes asolution having the same tonicity as some other solution with which itis compared, such as physiological salt solution or serum. Tonicityagents may be used in an amount of about 5 mM to about 350 mM, e.g., inan amount of 100 mM to 350 nM.

A surfactant may also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include polyoxyethylensorbitan fatty acid esters (Tween),polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitablepolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (soldunder the trademark Tween 20™) and polysorbate 80 (sold under thetrademark Tween 80™). Examples of suitable polyethylene-polypropylenecopolymers are those sold under the names Pluronic® F68 or Poloxamer188™. Examples of suitable Polyoxyethylene alkyl ethers are those soldunder the trademark Brij™. Exemplary concentrations of surfactant mayrange from about 0.001% to about 1% w/v.

A lyoprotectant may also be added in order to protect the labile activeingredient (e.g. a protein) against destabilizing conditions during thelyophilization process. For example, known lyoprotectants include sugars(including glucose and sucrose); polyols (including mannitol, sorbitoland glycerol); and amino acids (including alanine, glycine and glutamicacid). Lyoprotectants can be included in an amount of about 10 mM to 500nM.

In some embodiments, a subject formulation includes a subject antibody,and one or more of the above-identified agents (e.g., a surfactant, abuffer, a stabilizer, a tonicity agent) and is essentially free of oneor more preservatives, such as ethanol, benzyl alcohol, phenol,m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkoniumchloride, and combinations thereof. In other embodiments, a preservativeis included in the formulation, e.g., at concentrations ranging fromabout 0.001 to about 2% (w/v).

For example, a subject formulation can be a liquid or lyophilizedformulation suitable for parenteral administration, and can comprise:about 1 mg/mL to about 200 mg/mL of a subject antibody; about 0.001% toabout 1% of at least one surfactant; about 1 mM to about 100 mM of abuffer; optionally about 10 mM to about 500 mM of a stabilizer; andabout 5 mM to about 305 mM of a tonicity agent; and has a pH of about4.0 to about 7.0.

As another example, a subject parenteral formulation is a liquid orlyophilized formulation comprising: about 1 mg/mL to about 200 mg/mL ofa subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mMSucrose; and has a pH of 5.5.

As another example, a subject parenteral formulation comprises alyophilized formulation comprising: 1) 15 mg/mL of a subject antibody;0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pHof 5.5; or 2) 75 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mML-histidine; and 250 mM sucrose; and has a pH of 5.5; or 3) 75 mg/mL ofa subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mMSucrose; and has a pH of 5.5; or 4) 75 mg/mL of a subject antibody;0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has apH of 5.5; or 6) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20mM L-histidine; and 250 mM trehalose; and has a pH of 5.5.

As another example, a subject parenteral formulation is a liquidformulation comprising: 1) 7.5 mg/mL of a subject antibody; 0.022% Tween20 w/v; 120 mM L-histidine; and 250 125 mM sucrose; and has a pH of 5.5;or 2) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mML-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg/mLof a subject antibody; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mMsucrose; and has a pH of 5.5; or 4) 37.5 mg/mL of a subject antibody;0.02% Tween 20 w/v; 10 mM L-histidine; 125 mM trehalose; and has a pH of5.5; or 5) 37.5 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 10 mML-histidine; and 125 mM trehalose; and has a pH of 5.5; or 6) 5 mg/mL ofa subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mMtrehalose; and has a pH of 5.5; or 7) 75 mg/mL of a subject antibody;0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol; and has a pHof 5.5; or 8) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mML histidine; and 140 mM sodium chloride; and has a pH of 5.5; or 9) 150mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and250 mM trehalose; and has a pH of 5.5; or 10) 150 mg/mL of a subjectantibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol;and has a pH of 5.5; or 11) 150 mg/mL of a subject antibody; 0.02% Tween20 w/v; 20 mM L-histidine; and 140 mM sodium chloride; and has a pH of5.5; or 12) 10 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 20 mML-histidine; and 40 mM sodium chloride; and has a pH of 5.5.

A subject antibody can be utilized in aerosol formulation to beadministered via inhalation. A subject antibody can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject antibody can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. A subject antibody can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise a subject antibody in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of an anti-Tauantibody of the present disclosure, calculated in an amount sufficientto produce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for a subjectantibody may depend on the particular antibody employed and the effectto be achieved, and the pharmacodynamics associated with each antibodyin the host.

Other modes of administration will also find use with a method of thepresent disclosure. For instance, a subject antibody can be formulatedin suppositories and, in some cases, aerosol and intranasalcompositions. For suppositories, the vehicle composition will includetraditional binders and carriers such as, polyalkylene glycols, ortriglycerides. Such suppositories may be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10% (w/w),e.g., about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed. The nasal formulations may also containpreservatives such as, but not limited to, chlorobutanol andbenzalkonium chloride. A surfactant may be present to enhance absorptionof the subject antibody by the nasal mucosa.

A subject antibody can be administered as an injectable formulation.Typically, injectable compositions are prepared as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared. The preparationmay also be emulsified or the antibody encapsulated in liposomevehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of a subject antibody adequate toachieve the desired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In some embodiments, a subject antibody is formulated in a controlledrelease formulation. Sustained-release preparations may be preparedusing methods well known in the art. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody in which the matrices arein the form of shaped articles, e.g. films or microcapsules. Examples ofsustained-release matrices include polyesters, copolymers of L-glutamicacid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,hydrogels, polylactides, degradable lactic acid-glycolic acid copolymersand poly-D-(−)-3-hydroxybutyric acid. Possible loss of biologicalactivity and possible changes in immunogenicity of antibodies comprisedin sustained-release preparations may be prevented by using appropriateadditives, by controlling moisture content and by developing specificpolymer matrix compositions.

Controlled release within the scope of the present disclosure can betaken to mean any one of a number of extended release dosage forms. Thefollowing terms may be considered to be substantially equivalent tocontrolled release, for the purposes of the present disclosure:continuous release, controlled release, delayed release, depot, gradualrelease, long-term release, programmed release, prolonged release,proportionate release, protracted release, repository, retard, slowrelease, spaced release, sustained release, time coat, timed release,delayed action, extended action, layered-time action, long acting,prolonged action, repeated action, slowing acting, sustained action,sustained-action medications, and extended release. Further discussionsof these terms may be found in Lesczek Krowczynski, Extended-ReleaseDosage Forms, 1987 (CRC Press, Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems.

Physical systems include, but are not limited to, reservoir systems withrate-controlling membranes, such as microencapsulation,macroencapsulation, and membrane systems; reservoir systems withoutrate-controlling membranes, such as hollow fibers, ultra microporouscellulose triacetate, and porous polymeric substrates and foams;monolithic systems, including those systems physically dissolved innon-porous, polymeric, or elastomeric matrices (e.g., nonerodible,erodible, environmental agent ingression, and degradable), and materialsphysically dispersed in non-porous, polymeric, or elastomeric matrices(e.g., nonerodible, erodible, environmental agent ingression, anddegradable); laminated structures, including reservoir layers chemicallysimilar or dissimilar to outer control layers; and other physicalmethods, such as osmotic pumps, or adsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release may be found in Agis F. Kydonieus, Controlled ReleaseTechnologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

There are a number of controlled release drug formulations that aredeveloped for oral administration. These include, but are not limitedto, osmotic pressure-controlled gastrointestinal delivery systems;hydrodynamic pressure-controlled gastrointestinal delivery systems;membrane permeation-controlled gastrointestinal delivery systems, whichinclude microporous membrane permeation-controlled gastrointestinaldelivery devices; gastric fluid-resistant intestine targetedcontrolled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems may be found in Yie W. Chien, Novel DrugDelivery Systems, 1992 (Marcel Dekker, Inc.).

Dosages

A suitable dosage can be determined by an attending physician or otherqualified medical personnel, based on various clinical factors. As iswell known in the medical arts, dosages for any one patient depend uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex of the patient, time, androute of administration, general health, and other drugs beingadministered concurrently. A subject antibody may be administered inamounts between 1 ng/kg body weight and 20 mg/kg body weight per dose,e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, it canalso be in the range of 1 μg to 10 mg per kilogram of body weight perminute.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific antibody, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

Routes of Administration

A subject antibody is administered to an individual using any availablemethod and route suitable for drug delivery, including in vivo and exvivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intrathecal,subcutaneous, intradermal, topical application, intravenous,intraarterial, rectal, nasal, oral, and other enteral and parenteralroutes of administration. Routes of administration may be combined, ifdesired, or adjusted depending upon the antibody and/or the desiredeffect. A subject antibody composition can be administered in a singledose or in multiple doses. In some embodiments, a subject antibodycomposition is administered orally. In some embodiments, a subjectantibody composition is administered via an inhalational route. In someembodiments, a subject antibody composition is administeredintranasally. In some embodiments, a subject antibody composition isadministered locally. In some embodiments, a subject antibodycomposition is administered intracranially. In some embodiments, asubject antibody composition is administered intravenously. In someembodiments, a subject antibody composition is administeredintrathecally.

An antibody of the present disclosure can be administered to a hostusing any available conventional methods and routes suitable fordelivery of conventional drugs, including systemic or localized routes.In general, routes of administration contemplated by the inventioninclude, but are not necessarily limited to, enteral, parenteral, orinhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intrathecal, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery of asubject antibody. Where systemic delivery is desired, administrationtypically involves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

A subject antibody can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as a tauopathy. As such,treatment also includes situations where the pathological condition, orat least symptoms associated therewith, are completely inhibited, e.g.prevented from happening, or stopped, e.g. terminated, such that thehost no longer suffers from the pathological condition, or at least thesymptoms that characterize the pathological condition.

In some embodiments, a subject antibody is administered by injectionand/or delivery, e.g., to a site in a brain artery or directly intobrain tissue. A subject antibody can also be administered directly to atarget site e.g., by biolistic delivery to the target site.

A variety of hosts (wherein the term “host” is used interchangeablyherein with the terms “subject,” “individual,” and “patient”) aretreatable according to the subject methods. Generally such hosts are“mammals” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, andrats), and primates (e.g., humans, chimpanzees, and monkeys). In someembodiments, the hosts will be humans.

Kits with unit doses of a subject antibody, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the antibody in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Detection Methods

The present disclosure provides in vitro methods of detecting a Taupolypeptide in a biological sample obtained from an individual; andmethods of detecting a Tau polypeptide in a living individual in vivo. Asubject in vitro detection method can be quantitative. Tau can thusserve as a biomarker for progression of a tauopathy, or response totreatment for a tauopathy.

The Tau polypeptide that is detected/quantitated can be: a) full-lengthTau; b) an N-terminal fragment of full-length Tau; c) total Tau, where“total Tau” can include full-length Tau of any isoform; d) free Tau,e.g., Tau that is not bound to a subject anti-tau antibody; and e) anyN-terminal Tau fragments that are present in a biological sample andthat display the epitope recognized by a subject anti-Tau antibody.Amino acid sequences of human full-length Tau are presented in FIGS.6A-D.

In some cases, a subject detection method can further comprisedetermining the level of Aβ40 and/or Aβ42 in a biological sample.Determination of the level of Aβ40 and/or Aβ42 in a biological samplecan be carried out using an immunological assay (e.g., an ELISA), e.g.,using antibody that binds Aβ40 and/or Aβ42.

Suitable biological samples include, e.g., cerebrospinal fluid, blood,plasma, serum, urine, and saliva.

An in vitro method of the present disclosure of detecting a Taupolypeptide in a biological sample obtained from an individual generallyinvolves: a) contacting the biological sample with an anti-Tau antibodyas described herein; and b) detecting binding of the antibody to Taupolypeptide present in the sample. In some cases, the anti-Tau antibodycomprises VH and/or VL CDRs of depicted in FIGS. 1A and 1B. In somecases, the anti-Tau antibody comprises VH and/or VL CDRs of FIGS. 2A and2B.

A detection method of the present disclosure can be used to determinewhether an individual has, or is at risk of developing, a tauopathy. Adetection method of the present disclosure can be used to determine thestage (severity) of a tauopathy. A detection method of the presentdisclosure can be used to determine a patient's response to a treatmentregimen for treating a tauopathy. A biological sample can be testedusing a subject detection method, where the biological sample isobtained from an individual suspected of having a tauopathy, anindividual who has been diagnosed as having a tauopathy, an individualwho has a genetic predisposition to developing a tauopathy, etc.

The present disclosure provides a method of diagnosing aneurodegenerative tauopathy in an individual. The method generallyinvolves (a) assessing the level of a Tau polypeptide in a biologicalsample obtained from the individual; and (b) comparing the level of theTau polypeptide to a reference, a standard, or a normal control valuethat indicates the level of Tau in normal control subjects. Asignificant difference between the level of Tau polypeptide in thebiological sample and the normal control value indicates that theindividual has a neurodegenerative tauopathy.

The present disclosure provides a method of monitoring the progressionof, or monitoring response to treatment for, a neurodegenerativetauopathy in an individual. The method generally involves comparing thelevel of a Tau polypeptide in a biological sample obtained from theindividual at a first time point with the level of a Tau polypeptide ina biological sample obtained from the individual at a second time point.A difference in the level of the Tau polypeptide in a biological sampleobtained from the individual at a second time point, compared to thelevel of the Tau polypeptide in a biological sample obtained from theindividual at a first time point, can provide an indication as to: i)whether the tauopathy is progressing or whether progression of thedisease has halted; and/or ii) how quickly the tauopathy is progressing;and/or iii) whether the individual is exhibiting a beneficial clinicalresponse to treatment with a drug or other treatment regimen fortreating the tauopathy.

The present disclosure provides a method of staging a tauopathy. Forexample, a subject method can provide for staging Alzheimer's disease.For example, the level of a Tau polypeptide in a biological sample(e.g., the CSF or other liquid biological sample) from a livingindividual can provide an indication as to the Braak stage of AD. Braakand Braak (1995) Neurobiol. Aging 16:271. For example, the level of aTau polypeptide in a biological sample from a living individual canprovide an indication as to whether the individual is in transentorhinalstages I-II of AD; limbic stages III-IV of AD; or neocortical stagesV-VI of AD.

The level of a Tau polypeptide in a biological sample can be assessed byany suitable method known in the art. Suitable methods include, but arenot limited to, a protein (“Western”) blot, immunoprecipitation,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),fluorescent activated cell sorting (FACS), two-dimensional gelelectrophoresis, mass spectroscopy (MS), matrix-assisted laserdesorption/ionization-time of flight-MS (MALDI-TOF), surface-enhancedlaser desorption ionization-time of flight (SELDI-TOF), high performanceliquid chromatography (HPLC), fast protein liquid chromatography (FPLC),multidimensional liquid chromatography (LC) followed by tandem massspectrometry (MS/MS), and laser densitometry.

The present disclosure provides a method of monitoring progression of atauopathy in an individual, where the method generally involves: a)determining a first level of a Tau polypeptide in a biological sampleobtained from the individual at a first time point; b) determining asecond level of a Tau polypeptide in a biological sample obtained fromthe individual at a second time point; and c) comparing the second levelof Tau with the first level of Tau. The determining steps can comprise:i) contacting the biological sample with a subject anti-Tau antibody;and ii) quantitating binding of the antibody to Tau polypeptide presentin the sample.

In some cases, the first time point is a time point before initiation ofa treatment regimen, and the second time point is a time point afterinitiation of a treatment regimen. Thus, the instant disclosure providesa method of monitoring response to treatment with an agent that treats atauopathy, where the method involves: a) determining a first level of aTau polypeptide in a biological sample obtained from the individual at afirst time point that is before treatment with an agent to treat atauopathy is initiated; b) determining a second level of a Taupolypeptide in a biological sample obtained from the individual at asecond time point that is after initiation of treatment with an agent totreat a tauopathy; and c) comparing the second level of Tau with thefirst level of Tau.

A subject method of monitoring progression of a tauopathy can also beapplied to methods of monitoring progression of a synucleinopathy, e.g.,Parkinson's disease (PD); dementia with Lewy Bodies (DLB); multiplesystem atrophy (MSA); etc. For example, progression of PD with dementia(PDD) can be monitored with a subject method.

In some tauopathies, the level of Tau increases with progression of thedisease. In other tauopathies, the level of Tau decreases withprogression of the disease. Thus, e.g., the level of Tau increases withprogression of AD; and decreases with progression of FTD.

A subject method can involve use of a kit or an assay device comprisinga subject anti-Tau antibody. The present disclosure provides kits andassay devices for carrying out a method as described herein. A subjectkit includes an anti-tau antibody of the present disclosure.

The anti-tau antibody can be immobilized on an insoluble support (e.g.,a test strip, a well of a multi-well plate, a bead (e.g., a magneticbead), etc.). Suitable supports are well known in the art and comprise,inter alia, commercially available column materials, polystyrene beads,latex beads, magnetic beads, colloid metal particles, glass and/orsilicon chips and surfaces, nitrocellulose strips, nylon membranes,sheets, wells of reaction trays (e.g., multi-well plates), plastictubes, etc. A solid support can comprise any of a variety of substances,including, e.g., glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amylose, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Suitablemethods for immobilizing a subject antibody onto a solid support arewell known and include, but are not limited to ionic, hydrophobic,covalent interactions and the like. Solid supports can be soluble orinsoluble, e.g., in aqueous solution. In some embodiments, a suitablesolid support is generally insoluble in an aqueous solution.

An anti-tau antibody of the present disclosure can comprise a detectablelabel. Where the antibody comprises a detectable label, a subject kitcan include one or more reagents for developing the detectable label. Alabeled antibody can comprise a label such as a chemiluminescent agent,a particulate label, a colorimetric agent, an energy transfer agent, anenzyme, a fluorescent agent, or a radioisotope. Suitable detectablelabels include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, orchemical means. Suitable detectable labels include, but are not limitedto, fluorescent labels (e.g., fluorescein isothiocyanate, texas red,rhodamine, a green fluorescent protein, a red fluorescent protein, ayellow fluorescent protein, and the like); radiolabels (e.g., ³H, ¹²⁵I,³⁵S, ¹⁴C, or ³²P); and enzymes (e.g., horse radish peroxidase, alkalinephosphatase, luciferase, and other enzymes that act on a substrate toproduce a product that can be detected by fluorometric, colorimetric, orspectrophotometric means).

A subject kit can further include one or more additional components,where suitable additional components include: 1) a positive control; 2)a buffer (e.g., a binding buffer; a wash buffer; etc.); 3) reagents foruse in generating a detectable signal; and the like. Other optionalcomponents of the kit include: a protease inhibitor; a detectable label;etc. The various components of the kit may be present in separatecontainers or certain compatible components may be pre-combined into asingle container, as desired.

In addition to above-mentioned components, a subject kit can includeinstructions for using the components of the kit to practice a subjectmethod. The instructions for practicing a subject method are generallyrecorded on a suitable recording medium. For example, the instructionsmay be printed on a substrate, such as paper or plastic, etc. As such,the instructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.compact disc-read only memory (CD-ROM), digital versatile disk (DVD),diskette, etc. In yet other embodiments, the actual instructions are notpresent in the kit, but means for obtaining the instructions from aremote source, e.g. via the internet, are provided. An example of thisembodiment is a kit that includes a web address where the instructionscan be viewed and/or from which the instructions can be downloaded. Aswith the instructions, this means for obtaining the instructions isrecorded on a suitable substrate.

An assay device can include a subject anti-Tau antibody immobilized on asolid substrate. The assay device can be in any of a variety of formats,e.g., a test strip, a dipstick; etc.

In Vivo Imaging

As discussed above, the present disclosure provides methods of detectinga Tau polypeptide in a living individual, e.g., by an in vivo imagingtechnique. For example, in one embodiment, in vivo imaging of a Taupolypeptide can be accomplished by positron emission tomography (PET),single photon emission tomography (SPECT), near infrared (NIR) opticalimaging, or magnetic resonance imaging (MRI). A subject anti-tauantibody is administered to an individual, and the presence and/or levelof the tau polypeptide is detected. The anti-tau antibody can comprise alabel suitable for use in PET, SPECT, NIR, or MRI. Such labels include acontrast agent or a radioisotope, where the contrast agent orradioisotope is one that is suitable for use in imaging, e.g., imagingprocedures carried out on humans, as described above. In some cases, theanti-Tau antibody comprises VH and/or VL CDRs of IPN001. In some cases,the anti-Tau antibody comprises VH and/or VL CDRs of IPN002. Theanti-Tau antibody can comprise one or more humanized framework regions,as described above.

Generating a Report

In some instances, a subject detection method comprises detecting a Taupolypeptide in a biological sample obtained from an individual; and,based on the level of detected Tau polypeptide, generating a reportand/or directing therapy or management of the individual from whom thebiological sample was obtained.

A report can include one or more of: an indication as to whether theindividual likely has a tauopathy; an indication of the severity of thetauopathy; an indication as to whether the individual exhibits abeneficial clinical response to treatment for the tauopathy; and thelike.

Thus, a report can include information such as a predicted likelihoodthat the individual has, or will develop, a tauopathy; a recommendationregarding further evaluation; a recommendation regarding therapeuticdrug and/or other health management intervention; and the like.

For example, the methods disclosed herein can further include a step ofgenerating or outputting a report providing the results of a subjectassessment, which report can be provided in the form of an electronicmedium (e.g., an electronic display on a computer monitor), or in theform of a tangible medium (e.g., a report printed on paper or othertangible medium). An assessment as to the likelihood that a person has,or at risk of developing, a tauopathy can be referred to as a “riskreport,” “a risk score,” or “a likelihood score.” A person or entitythat prepares a report (“report generator”) may also perform steps suchas sample gathering, sample processing, and the like. Alternatively, anentity other than the report generator can perform steps such as samplegathering, sample processing, and the like. A risk assessment report canbe provided to a user. A “user” can be a health professional (e.g., aclinician, a laboratory technician, or a physician).

Directing Health Management

In some instances, a subject detection method comprises detecting a Taupolypeptide in a biological sample obtained from an individual; and,based on the level of detected Tau polypeptide, generating a reportand/or directing therapy or management of the individual from whom thebiological sample was obtained.

Thus, e.g., depending on the outcome of a subject detection method, arecommendation can be made that the individual undergo therapeuticintervention (treatment) for the tauopathy and/or that the individual beconsidered for special health management.

Therapeutic intervention can include, e.g., drug therapy for thetreatment of Alzheimer's disease. Examples of drug therapy for thetreatment of Alzheimer's disease include, but are not limited to,acetylcholinesterase inhibitors, including, but not limited to, Aricept(donepezil), Exelon (rivastigmine), metrifonate, and tacrine (Cognex);an anti-Aβ antibody (e.g., solanezumab); an anti-tau antibody;non-steroidal anti-inflammatory agents, including, but not limited to,ibuprofen and indomethacin; cyclooxygenase-2 (Cox2) inhibitors such asCelebrex; and monoamine oxidase inhibitors, such as Selegilene (Eldeprylor Deprenyl). Dosages for each of the above agents are known in the art.For example, Aricept can be administered at 50 mg orally per day for 6weeks, and, if well tolerated by the individual, at 10 mg per daythereafter.

Determining the Amount of Free and Bound Extracellular Tau

Following administration of an anti-eTau antibody to an individual, itmay be of interest to determine the amount of eTau remaining in the CSFor ISF that is not bound to the anti-eTau antibody. The presentdisclosure provides methods for determining the amount of such freeeTau. A schematic representation of a method for determining the amountof eTau remaining in the CSF or ISF that is not bound to the anti-eTauantibody is depicted in FIG. 54A. Following administration of ananti-eTau antibody to an individual, it may also be of interest todetermine the amount of eTau in CSF or ISF that is bound to an anti-eTauantibody. A schematic representation of a method for determining theamount of eTau in CSF or ISF that is bound to an anti-eTau antibody isdepicted in FIG. 54B.

Determining the Amount of Free Extracellular Tau

The present disclosure provides a method of determining the amount ofextracellular Tau (eTau) unbound to an anti-eTau antibody in a sample ofCSF or ISF obtained from a subject undergoing therapy with the anti-eTauantibody. The method generally involves: a) contacting an immobilizedantibody with a sample of CSF or ISF obtained from the subject, wherethe immobilized antibody competes for binding to eTau with the anti-eTauantibody administered to the subject, and where the contacting is underconditions suitable for binding of the unbound eTau to the immobilizedantibody; and b) determining the amount of eTau bound to the immobilizedantibody. The amount of eTau bound to the immobilized antibody is anindication of the amount of eTau unbound to the anti-Tau antibody in thesample. In some cases, the amount of eTau bound to the immobilizedantibody is determined using a detectably labeled third antibody thatdoes not compete with the immobilized antibody for binding to the eTau.

As noted above, the assay measures the amount of eTau in a CSF or ISFsample obtained from an individual undergoing therapy with an anti-eTauantibody. In some cases, the anti-eTau antibody is a therapeutichumanized anti-eTau antibody. In some cases, the anti-eTau antibody is ahumanized anti-eTau antibody of the present disclosure. In some cases,the anti-eTau antibody is hu-IPN002.

In some cases, a subject method will comprise: a) determining the amountof eTau unbound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and b) determining the level of total tau in thesample.

In some cases, a subject method will comprise: a) determining the amountof eTau unbound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and b) comparing the level of unbound Tau in thesample to the level of total tau in a CSF or ISF sample obtained fromthe individual before treatment with the anti-eTau antibody.

A subject detection method is suitable for determining the level ofextracellular tau. “Extracellular tau” (“eTau”), as used herein,encompasses any Tau polypeptide that can be detected in cerebrospinalfluid (CSF) or interstitial fluid (ISF). In some embodiments, eTau is apolypeptide having a length of 175 amino acids and comprising aminoacids 2-176 of full-length tau; for example, in some embodiments eTau isa polypeptide comprising the amino acid sequence set forth in SEQ IDNO:45. In some embodiments, eTau is a polypeptide having a length of 171amino acids and comprising amino acids 2-172 of full-length tau; forexample, in some embodiments eTau is a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:44. In some embodiments, eTau is aneTau-2 polypeptide comprising the amino acid sequence set forth in SEQID NO:46. In some embodiments, eTau is an eTau-3 polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO:47. In some embodiments,eTau is an eTau-4 polypeptide comprising the amino acid sequence setforth in SEQ ID NO:48.

In some cases, an eTau polypeptide has a length of from about 50 aminoacids to about 175 amino acids, e.g., from about 50 amino acids (aa) toabout 75 aa, from about 75 aa to about 100 aa, from about 100 aa toabout 125 aa, from about 125 aa to about 150 aa, or from about 150 aa toabout 175 aa; and can comprise from 50 to about 75, from about 75 toabout 100, from about 100 to about 125, from about 125 to about 150, orfrom about 150 to about 175, contiguous amino acids of amino acids 2-176of full-length tau. Exemplary eTau polypeptides are depicted in FIG. 20.

Determining the Amount of Extracellular Tau Bound to an Anti-eTauAntibody

The present disclosure provides a method of determining the amount ofeTau bound to a therapeutic anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the therapeuticanti-eTau antibody. The method generally involves: a) contacting animmobilized antibody with a sample of CSF or ISF obtained from thesubject, where the immobilized antibody does not compete for binding toeTau with the anti-eTau antibody administered to the subject, saidcontacting being under conditions suitable for binding of thetherapeutic antibody-bound eTau to the immobilized antibody; and b)determining the amount of therapeutic anti-eTau/eTau complex bound tothe immobilized antibody, wherein the amount of therapeutic anti-eTauantibody/eTau complex bound to the immobilized antibody is an indicationof the amount of therapeutic antibody-bound eTau present in the sample.The amount of therapeutic anti-eTau antibody/eTau complex bound to theimmobilized antibody is determined by detecting the anti-eTau antibodypresent in the anti-eTau antibody/eTau complex. The amount oftherapeutic anti-eTau antibody/eTau complex bound to the immobilizedantibody is determined by detecting the anti-eTau antibody present inthe anti-eTau antibody/eTau complex provides an indication of the amountof Tau bound to the therapeutic antibody in the CSF or ISF sample.

As noted above, the assay measures the amount of therapeuticantibody-bound eTau in a CSF or ISF sample obtained from an individualundergoing therapy with the anti-eTau antibody. In some cases, theanti-eTau antibody is a therapeutic humanized anti-eTau antibody. Insome cases, the anti-eTau antibody is a humanized anti-eTau antibody ofthe present disclosure. In some cases, the anti-eTau antibody ishu-IPN002.

In some cases, a subject method will comprise: a) determining the amountof eTau bound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and b) determining the level of total tau in thesample. In some cases, a subject method will comprise: a) determiningthe amount of eTau bound to an anti-eTau antibody in a sample of CSF orISF obtained from a subject undergoing therapy with the anti-eTauantibody, as described above; and b) determining the amount of eTau inthe CSF or ISF sample that is not bound to the therapeutic anti-eTauantibody. In some cases, a subject method will comprise: a) determiningthe amount of eTau bound to an anti-eTau antibody in a sample of CSF orISF obtained from a subject undergoing therapy with the anti-eTauantibody, as described above; b) determining the level of total tau inthe sample; and c) determining the amount of eTau in the CSF or ISFsample that is not bound to the therapeutic anti-eTau antibody.

In some cases, a subject method will comprise: a) determining the amountof eTau bound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and b) comparing the level of anti-e-Tauantibody-bound Tau in the sample to the level of total tau in a CSF orISF sample obtained from the individual before treatment with theanti-eTau antibody.

In some cases, a subject method will comprise: a) determining the amountof eTau unbound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; b) determining the amount of eTau bound to ananti-eTau antibody in a sample of CSF or ISF obtained from a subjectundergoing therapy with the anti-eTau antibody, as described above; andc) comparing the level of unbound Tau and the level of anti-eTauantibody-bound Tau in the sample to the level of total tau in a CSF orISF sample obtained from the individual before treatment with theanti-eTau antibody.

A subject detection method is suitable for determining the level ofextracellular tau bound to a therapeutic antibody. “Extracellular tau”(“eTau”), as used herein, encompasses any Tau polypeptide that can bedetected in cerebrospinal fluid (CSF) or interstitial fluid (ISF). Insome embodiments, eTau is a polypeptide having a length of 175 aminoacids and comprising amino acids 2-176 of full-length tau; for example,in some embodiments eTau is a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:45. In some embodiments, eTau is apolypeptide having a length of 171 amino acids and comprising aminoacids 2-172 of full-length tau; for example, in some embodiments eTau isa polypeptide comprising the amino acid sequence set forth in SEQ IDNO:44. In some embodiments, eTau is an eTau-2 polypeptide comprising theamino acid sequence set forth in SEQ ID NO:46. In some embodiments, eTauis an eTau-3 polypeptide comprising the amino acid sequence set forth inSEQ ID NO:47. In some embodiments, eTau is an eTau-4 polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:48.

In some cases, an eTau polypeptide has a length of from about 50 aminoacids to about 175 amino acids, e.g., from about 50 amino acids (aa) toabout 75 aa, from about 75 aa to about 100 aa, from about 100 aa toabout 125 aa, from about 125 aa to about 150 aa, or from about 150 aa toabout 175 aa; and can comprise from 50 to about 75, from about 75 toabout 100, from about 100 to about 125, from about 125 to about 150, orfrom about 150 to about 175, contiguous amino acids of amino acids 2-176of full-length tau. Exemplary eTau polypeptides are depicted in FIG. 20.

Generating a Report

In some cases, a subject method will comprise: a) determining the amountof eTau unbound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and b) generating a report and/or directing therapyor management of the individual from whom the sample was obtained. Insome cases, a subject method will comprise: a) determining the amount ofeTau unbound to an anti-eTau antibody in a sample of CSF or ISF obtainedfrom a subject undergoing therapy with the anti-eTau antibody, asdescribed above; b) comparing the level of unbound Tau in the sample tothe level of total tau in a CSF or ISF sample obtained from theindividual before treatment with the anti-eTau antibody; and c)generating a report and/or directing therapy or management of theindividual from whom the sample was obtained.

In some cases, a subject method will comprise: a) determining the amountof eTau bound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and b) generating a report with the results of thedetermination. The report can further include one or both of the amountof unbound Tau in the sample to the level of total tau in a CSF or ISFsample after treatment with the anti-Tau antibody; and the amount oftotal Tau in a CSF or ISF sample obtained from the individual beforetreatment with the anti-eTau antibody.

A report can include, e.g., an indication as to whether the individualexhibits a beneficial clinical response to treatment for the tauopathy;an indication as to whether the dosage of the anti-eTau antibody shouldbe maintained, increased, or decreased; and the like.

Thus, a report can include information such as a recommendationregarding further evaluation; a recommendation regarding therapeuticdrug and/or other health management intervention; a recommendation toincrease the dosage of anti-eTau antibody; a recommendation to maintainthe dosage of anti-eTau antibody; a recommendation to reduce the dosageof anti-eTau antibody; and the like.

For example, the methods disclosed herein can further include a step ofgenerating or outputting a report providing the results of a subjectassessment, which report can be provided in the form of an electronicmedium (e.g., an electronic display on a computer monitor), or in theform of a tangible medium (e.g., a report printed on paper or othertangible medium). A person or entity that prepares a report (“reportgenerator”) may also perform steps such as sample gathering, sampleprocessing, and the like. Alternatively, an entity other than the reportgenerator can perform steps such as sample gathering, sample processing,and the like. A risk assessment report can be provided to a user. A“user” can be a health professional (e.g., a clinician, a laboratorytechnician, or a physician).

Directing Health Management

In some instances, a subject method comprises a) determining the amountof eTau unbound to an anti-eTau antibody in a sample of CSF or ISFobtained from a subject undergoing therapy with the anti-eTau antibody,as described above; and, based on the determined amount of eTau unboundto an anti-eTau antibody, generating a report and/or directing therapyor management of the individual from whom the biological sample wasobtained.

In some cases, a subject method comprises a) determining the amount ofeTau unbound to an anti-eTau antibody in a sample of CSF or ISF obtainedfrom a subject undergoing therapy with the anti-eTau antibody, asdescribed above; and, based on the determined amount of eTau unbound toan anti-eTau antibody, maintaining the dosage of anti-eTau antibody thatwas administered to the subject. In some cases, a subject methodcomprises a) determining the amount of eTau unbound to an anti-eTauantibody in a sample of CSF or ISF obtained from a subject undergoingtherapy with the anti-eTau antibody, as described above; and, based onthe determined amount of eTau unbound to an anti-eTau antibody,increasing the dosage of anti-eTau antibody administered to the subject.In some cases, a subject method comprises a) determining the amount ofeTau unbound to an anti-eTau antibody in a sample of CSF or ISF obtainedfrom a subject undergoing therapy with the anti-eTau antibody, asdescribed above; and, based on the determined amount of eTau unbound toan anti-eTau antibody, decreasing the dosage of anti-eTau antibodyadministered to the subject.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Cloning and Sequencing of IPN001 and IPN002 VH and VL Regions

Amino acid sequences of the VH and VL regions of IPN001 (also referredto herein as “IPN1” or “IPN-1”) and IPN002 (also referred to herein as“IPN2” or “IPN-2”) antibodies were determined. The amino acid sequencesof the VH and VL regions of IPN001 are depicted in FIGS. 1A and 1B,respectively. The amino acid sequences of the VH and VL regions ofIPN002 are depicted in FIGS. 2A and 2B, respectively. The CDRs are inbold text and are underlined. CDRs were determined using the method ofKabat et al. (see Table 1; and J. Biol. Chem. 252:6609-6616 (1977); andKabat et al., U.S. Dept. of Health and Human Services, “Sequences ofproteins of immunological interest” (1991)).

Example 2: Electrophysiological Analysis of the Effect of Anti-TauAntibodies Materials and Methods

Whole cell patch clamp recording from induced pluripotent stem cells(iPSC) derived cortical neurons cultured on a monolayer of normal humanastrocytes was carried out using a patch pipette (2-5 MOhm) filled withsolution containing (mM): K-methyl-sulfate (140), NaCl (10), CaCl₂ (1),Mg-ATP (3); Na-GTP (0.4), EGTA (0.2), HEPES (10), Phosphocreatine withadjusted pH=7.3 and mOsm=300. Neurons were perfused (2 ml/min) withartificial cerebral spinal fluid containing (mM): NaCl (140), KCl (2.5),MgCl₂ (2) CaCl₂ (2), Hepes (10), D-Glucose (10), sucrose (20). AdjustedpH=7.4 mOsm=310. Recordings were done using pClamp-10.3 data acquisitionsoftware (Molecular Devices) and MultiClamp 700B amplifier (AxonInstrument; Foster City Calif.). AD tau and AD Tau pre-incubated withIPN001 or IPN002 (2-hrs at room temperature or 24-hrs at 4 degree C. at10:1 weight ratio) were applied via MinisQuirt micro-perfusion system(AutoMate, Berkeley, Calif.). Data analysis was done off-line usingClampfit 10.2 analysis software (Molecular Devices). All recordings weredone at room temperature.

Results

The data are depicted in FIGS. 3A-D.

Application of AD-Tau (6 μg/ml) causes cortical neuron membranedepolarization (A, B, and C). Pre-incubation of AD-Tau (6 μg/ml) withIPN001 (60 μg/ml) (A) or IPN002 (60 μg/ml) (B) for >2 hrs reduces AD-Taumediated membrane depolarization. C. Pre-incubation of AD-Tau (6 μg/ml)with mouse IgG (60 μg/ml) did not reduce AD-Tau mediated membranedepolarization in cortical neurons. D. Data summary showing IPN001 andIPN002 significantly reduced AD-Tau mediated membrane depolarization(Paired t-test *p<0.037; **p<0.009, p<0.003).

Example 3: Immunoreactivity of IPN001 and IPN002 with Tau in CSF from ADPatients

Cerebrospinal fluid (CSF) was pooled from 10 healthy donors (1 ml each).CSF was also pooled from 10 Alzheimer's disease (AD) patients (1 mleach). Aliquots of CSF pools were saved for ELISA analysis. 10 mls ofconditioned media from cortical neurons differentiated for 315 days froma Down's induced pluripotent stem cell (iPSC) iPSC line (8941.1) wasused as a control for the CSF affinity isolations and an aliquot wasalso saved for ELISA analysis. To determine if CSF containsIPN002-reactive Tau, each of the CSF pooled samples and conditionedmedia were precleared on an IgG1 coupled resin and the flow-throughsubsequently applied to an IPN001-coupled resin. The IPN001 resins werewashed thoroughly with phosphate buffered saline (PBS) and boundproteins eluted with 50 mM Glycine, 150 mM NaCl, pH 2.3 and neutralizedwith 1M Tris, pH 8.3 after elution. The eluted proteins wereconcentrated on YM10 concentrators and added to sample buffer for sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)electrophoresis and Western blotting analysis.

To determine if IPN002 reacts with any form of Tau in the CSF, Westernblots of the IPN002 eluted protein were probed with IPN001, Santa CruzTau H-150 (aa1-150) and with Dako's Tau #A0024 antibody which reactswith the C-termini (aa#243-441) of Tau. The data are depicted in FIGS.4A-C.

Western blots showed IPN001 (FIG. 4a ) and Tau H-150 (FIG. 4b )immunoreactive bands present in the IPN002 affinity purified proteinfrom both healthy and AD CSF that ranged in molecular weight from ˜25 kdto 37 kd. These Tau fragments were similar in their sizes, but not intheir relative abundance, to eTau fragments isolated from the Down'sline conditioned media. The Dako C-terminal tau antibody (FIG. 4c ) didnot detect any reactive species from the IPN002 affinity isolation fromeither CSF or from conditioned media. Full-length Tau was not detectedby any of the Tau antibodies from the IPN002 affinity isolation. Becausethe IPN002 affinity isolated proteins were reactive with IPN001 andIPN002 on Western blot, it was concluded that Tau in CSF is also IPN001reactive.

The CSF and conditioned media flow-through from the IPN002 affinityresins were then applied sequentially to and eluted from T46 (Tau#428-441) and HT7 (Tau #159-163) to determine if any C-terminal ormid-region tau fragments were present that were not isolated by IPN002.The eluates were probed with the Dako C-terminal antibody (FIG. 4c ) butno immunoreactivity was detected. These data suggest that IPN001 andIPN002 immunoreactive tau is more abundant than full length, mid-regiononly or C-terminal tau fragments.

Aliquots of the flow-through from each of the CSFs and conditioned mediawere saved for pre- versus post-isolation comparison to determine if alldetectable tau was removed during the isolation, using a commerciallyavailable kit commonly used to determine Tau levels in CSF. The data areshown in FIG. 5. This analysis demonstrated that all the detectable tauwas removed from the post-CSF samples during the affinity isolationprocess.

These data provide strong evidence that both IPN001 and IPN002 reactwith the major tau species present in CSF from both healthy and ADpatients.

Example 4: Detection of eTau in Patient Samples Materials and Methods

Conditioned Media Collection from iPSC-Derived Cortical Neurons

iPSC (induced pluripotent stem cells) were generated from healthy agematched controls and Alzheimer's patients using the Yamanaka method(Takahashi et al. (2007) Cell 131(5), 861) as described in Dimos et al.(2008) Science 321:1218. iPSC were differentiated to cortical neuronslargely in line with published protocols using the dual SMAD monolayermethod (Chambers et al. (2009) Nat. Biotechnol. 27:275) followed bycortical neuron differentiation similar to that described in Shi et al.(2012) Nat. Neurosci. 15:477). iPSC-derived cortical neurons (iPSC-CN),cultured for 108 days, were washed, fresh media added, and conditionedmedia collected after three days unless otherwise noted. Multipledifferentiations from the lines were conducted to ensure reproducibilityof the eTau levels. Conditioned media was spun at 15,000 rpm for 15minutes prior to processing for Western blot or tau ELISA. For thebrefeldin A experiment, iPSC-CN cultures were washed with PBS prior toaddition of fresh media with and without 1 μM brefeldin A and mediaconditioned for one hour prior to collection.

Conditioned Media Collection from Human Primary Cortical Neurons

Human cortical neuron cultures (HCC) were prepared as described inWright et al. (2007) Neurobiol. Aging 28:226. Briefly, human fetalcerebral cortical tissue was obtained by Advanced Bioscience Resources(Alameda, Calif.) and complied with federal guidelines for fetalresearch and with the Uniformed Anatomical Gift Act. The tissue wasrinsed in Hank's buffered saline solution (Cellgro) and triturated inthe presence of 1 μg/ml DNase (EMD) and passed through a 100 μm cellstrainer. After centrifugation the pellet was resuspended in 0.05%trypsin/EDTA (Invitrogen) for 20 min at 37° C. Trypsin was inactivatedby adding an equal volume of media containing 10% fetal bovine serum(FBS) and sample gently triturated again in presence of DNase. Aftercentrifugation, cells were resuspended in plating media (Neurobasalcontaining B27, Invitrogen) and counted. Cells were plated in plates oron coverslips coated with poly-d-lysine with laminin. Three week old HCCwere washed, fresh media added and media collected after three days ofconditioning. Conditioned media was spun at 15,000 rpm for 15 minutesprior to processing for Western blot.

P301L Mouse ISF and Human CSF Collections

Mice were anesthetized using isoflurane (2%, 800 mL/min O₂).Bupivacain/epinephrine was used for local analgesia and fynadine orcarprophen for peri-/post-operative analgesia. The animals were placedin a stereotaxic frame (Kopf instruments, USA). Push-pull microdialysisprobes (phosphatidyl ethanolamine (PEE) membrane, Brainlink, theNetherlands) were inserted into the hippocampus (3 mm exposed surface).Microdialysis sampling was performed 24 and 48 hours after surgery. Onthe days of the sampling, the probes of the animals were connected withfluorinated ethylene propylene (FEP) tubing to a microperfusion pump(Harvard PHD 2000 Syringe pump, Holliston, Mass. or similar).Microdialysis probes were perfused with artificial CSF (aCSF) containing147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl₂ and 1.2 mM MgCl₂, and 0.15% bovineserum albumin (BSA) at a flow rate of 0.75 μL/min. Microdialysis sampleswere collected for 60 minute periods. After the stabilization period,basal samples were collected. On the second day of sampling, the aboveprocedure was repeated (Brains Online). The interstitial fluid (ISF) wasspun at 15,000 rpm for 15 minutes and cleared supernatants used for eTauWestern blots.

10 mls of CSF (Precision Med) from a pool of 10 healthy (Precision Med),10 AD patients (Precision Med) and 10 PSP patients were collected, spunat 15,000 rpm for 15 minutes, supernatants precleared on IgG affinityresin followed by tau isolation on an IPN002 anti-tau affinity resin,washed, eluted with 50 mM glycine, pH 2.3 with 150 mM NaCl into a tubecontaining 1M TBS, pH 8.3 to neutralize the pH, concentrated on YM10filters and prepared for tau Western blots. iPSC-CN conditioned mediafrom a fAD PSEN1 patient was similarly isolated as a positive control tocompare banding patterns.

Western Blots

Conditioned media were diluted in Laemmli buffer (Sigma). Culturedneurons were rinsed with PBS before incubation in 0.05% trypsin in DMEM(Invitrogen), rinsed and lysed in Laemmli buffer. All samples wereboiled, separated on tris-glycine polyacrylamide gels (Invitrogen) andtransferred to nitrocellulose using iBlot (Invitrogen). Membranes wereincubated in blocking buffer (LiCor), probed with 0.5 μg/ml IPN001antibody to tau and antibody to β-actin (1:2000; Abcam) in blockingbuffer containing 0.1% Tween-20, and anti-mouse 680 and anti-rabbit 800secondary antibodies (LiCor). Blots were scanned with the Odyssey SAinfrared imaging system and analyzed using Odyssey SA software (LiCor).

Tau ELISA

Media were collected after a three day conditioning period fromiPSC-derived cortical neuron cultures and assayed using an Alphascreenhomogeneous assay to measure tau. 10 μg/ml anti-tau AlphaLISA acceptorbeads and 1 nM biotinylated-anti-tau antibody were mixed withconditioned media overnight at room temperature. 40 μg/mlstreptavidin-donor beads (Perkin Elmer) were added for 30 minutes atroom temperature and the plate read on Envision plate reader.

eTau Purification

Conditioned media collected from iPSC-CN from AD patients was spun at15,000 rpm for 15 minutes, supernatants collected and precleared on anIgG affinity resin. The precleared supernatant was passed through anIPN002 anti-tau antibody resin, washed and eTau eluted with 50 mM sodiumcitrate, pH 2.3 with 150 mM NaCl into a tube containing 1M TBS, pH 8.3to neutralize the pH. The eluate was concentrated and buffer exchangedto PBS.

Immunofluorescence

MCC were rinsed with PBS, fixed in 4% paraformaldehyde, blocked with 10%normal donkey serum (Jackson ImmunoResearch) in PBS, permeabilized(unless otherwise specified) with 0.2% Triton-x-100 in PBS for 15minutes, and stained using IPN001 antibody to tau withdonkey-anti-mouse-A488 secondary antibody (Molecular Probes) and DAPI(Invitrogen). Images were acquired using the Leica DMI 600 B microscopeat 40× using the LAS AF software (Leica). Confocal images were acquiredusing the Nikon Eclipse Ti confocal microscope (Nikon).

Results

Assays were conducted to detect eTau fragments in various fluids. Theresults are depicted in FIG. 7. As shown in FIG. 7, left panel,endogenous tau is secreted from cortical neurons derived from humaninduced pluripotent stem cells (human iPSC-cortical neurons; iPSC-CN),where the secreted Tau is referred to as extracellular Tau or “eTau.” Asshown in FIG. 7, second panel from left, eTau is also present inconditioned media from human primary neurons (human cortical cells;“HCC”), confirming that eTau is not an artifact of iPSC-differentiation.These eTau fragments were also detected in neuronal lysates, suggestingthat tau is cleaved inside neurons prior to eTau secretion.

As shown in FIG. 7, middle panel, similar tau fragments were detected ininterstitial fluid (ISF) from P301L tau mice, where full length tau wasnot detected in either system. P301L mice are transgenic for a form ofhuman tau having a P301L mutation; P301L mice are models for humantauopathy. See, e.g., Gotz et al. (2001) J. Biol. Chem. 276:529; andLewis et al. (2000) Nature Genetics 25:402.

As shown in FIG. 7, right panels, eTau levels are increased in CSF fromAD patients, and in multiple lines from familial AD (fAD) patientscompared to lines from healthy patients. As shown in FIG. 7, rightpanels, eTau was also detected in CSF from PSP patients.

Example 5: eTau Induces Neuronal Hyperactivity Methods

Whole cell patch clamp recording from iPSC-CN cultured on monolayer ofnormal human astrocytes using micro-pipette (2-5 MOhm) were filled withsolution containing (mM): K-methyl-sulfate (140), NaCl (10), CaCl₂ (1),Mg-ATP (3); Na-GTP (0.4), EGTA (0.2), HEPES (10), Phosphocreatine (10)with adjusted pH=7.3, and mOsm=305. Neurons were perfused (2 ml/min)with artificial cerebral spinal fluid containing (mM): NaCl (140), KCl(2.5), MgCl₂ (2) CaCl₂ (2), Hepes (10), D-Glucose (10), sucrose (20),adjusted pH=7.4 mOsm=310. Recordings were made using pClamp-10.3 dataacquisition software (Molecular Devices) and MultiClamp 700B amplifier(Axon Instrument; Foster City Calif.). Puff application of eTau, or eTauwith inhibitors, tetrodotoxin (TTX) (Tocris), MK801 (Sigma), NBQX(Tocris), or anti-tau antibody, IPN001, was performed using MiniSquirtmicro-perfusion system (AutoMate, Berkeley, Calif.). Off-line dataanalysis used Clampfit 10.2 analysis software (Molecular Devices).Recordings were conducted at 34-37° C.

Results

To determine whether eTau can alter neuronal function, purified eTaufragment eTau was applied to iPSC-CN or HCC. The results are shown inFIGS. 8A-C.

As shown in FIG. 8A, addition of a purified eTau fragment mixture ontothese neurons promoted hyperactivity. As shown in FIG. 8B, hyperactivityinduced by the eTau mixture was inhibited by tetrodotoxin (TTX) and bythe NMDA and AMPA glutamate receptor antagonists, MK801 and NBQX,respectively. TTX blocks action potentials in nerves by binding to thevoltage-gated, fast sodium channels in nerve cell membranes. These datasuggest that eTau-induced neuronal hyperactivity is dependent on actionpotential-mediated release of glutamate. In contrast, as shown in themiddle panel of FIG. 8A, application of full length tau produced nodetectable changes in neuronal activity even at substantially higherconcentrations, showing that eTau-induced hyperactivity is dependent ontau fragments. These eTau-induced hyperactivity results strongly suggestthat calcium mobilization could be occurring in the neurons. Todetermine whether calcium mobilization occurs in the neurons, the effectof eTau on calcium mobilization was tested. As shown in FIG. 8C, eTau-1arobustly mobilized calcium. This type of neuronal hyperactivity, ifsustained in a chronic setting such as in AD, could result in neuronaldysfunction through altered synaptic firing and aberrant neuronalstimulation. eTau-1a includes amino acids 2-166 of fetal tau, i.e.,amino acids 2-166 of SEQ ID NO:27.

Example 6: Anti-Tau Antibody Reduces eTau-Mediated NeuronalHyperactivity

Electrophysiological analyses were carried out as described in Example5. The effect of IPN001 and IPN002 on e-Tau-mediated neuronalhyperactivity was assessed.

As shown in FIG. 8D, IPN001 reduces eTau-mediated neuronalhyperactivity. As shown in FIG. 19B, IPN002 reduces eTau-mediatedneuronal hyperactivity.

Example 7: Humanized Anti-Tau Antibodies

Humanized variants of IPN002 were generated. Amino acid sequences of theheavy chain VH domains of humanized variants 1-4, and nucleotidesequences encoding the heavy chain VH domain of the humanized variants,are shown in FIGS. 9-12. Amino acid sequences of the light chain VLdomain of humanized variants 1-4, and nucleotide sequences encoding thelight chain VL domain of the humanized variants, are shown in FIGS.13-16. Amino acid differences relative to the amino acid sequence ofIPN002 are summarized in Tables 4 and 5.

TABLE 4 VH Variants IPN002 Amino Acid (Parental VH Vari- VH Vari- VHVari- VH Vari- Position antibody) ant 1 ant 2 ant 3 ant 4 3 H H H Q Q 19K R R R R 40 T A A A A 42 D G G G G 44 R G G G G 66 Q R R R R 83 S S N NN 85 L S L L L 86 K K R R R 87 S S A A A 93 S S S S A 108 S S T T T

TABLE 5 Vk Variants IPN002 Amino Acid (Parental Vk Vari- Vk Vari- VkVari- Vk Vari- Position antibody) ant 1 ant 2 ant 3 ant 4 3 L L V V V 7T S S S S 14 S T T T T 17 D Q Q Q Q 18 Q P P P P 45 K Q Q Q Q 48 V V V VI 83 L V V V V 85 T T T V V 104 L V V V V

Single letter amino acid codes are as follows:

G—Glycine (Gly) P—Proline (Pro) A—Alanine (Ala) V—Valine (Val) L—Leucine(Leu) I—Isoleucine (Ile) M—Methionine (Met) C—Cysteine (Cys)F—Phenylalanine (Phe) Y—Tyrosine (Tyr) W—Tryptophan (Trp) H—Histidine(His) K—Lysine (Lys) R—Arginine (Arg) Q—Glutamine (Gln) N—Asparagine(Asn) E—Glutamic Acid (Glu) D—Aspartic Acid (Asp) S—Serine (Ser)T—Threonine (Thr) Example 8: Characterization of Humanized IPN002Variants

The relative tau binding affinities for binding to each of therecombinant tau (383 amino acid recombinant tau) as well as to eTau 1a,eTau1b, eTau2, eTau3 and eTau4 for each of the 16 antibody combinationsof VH#1-4 with Vk#1-4 are shown in Table 4, which is presented in FIG.17. The relative binding affinities for each tau and eTau species rangefrom 121 pM to 1030 pM for each of the VH/Vk antibody combinations. eTau1a includes amino acids 2-166 of fetal tau, i.e., amino acids 2-166 ofSEQ ID NO:27; and eTau 1b includes amino acids 2-196 and 217-228 offetal tau, i.e., amino acids 2-196 and 217-228 of SEQ ID NO:27. Aminoacid sequences of eTau3 and eTau 4 are depicted in FIG. 20.

To obtain absolute affinities as well as K_(on) and K_(dis) for theseVH/Vk human antibodies, Octet analysis was conducted using tau (383amino acid recombinant tau). The K_(D)'S ranged from 42.6 pM to 2120 pM.For all VH/Vk variants, the K_(on) values were high, and K_(dis) valueswere low for Tau and for each eTau species. The data are provided inTable 5, which is presented in FIG. 18.

A subset of the above-described humanized IPN002 variants was tested inadditional analysis. As shown in FIG. 19A, three variants, VH2/Vk1,VH2/Vk2, and VH2/Vk3, were used in a Western blot assay with a varietyfor samples containing tau. The tau-containing samples included iPSC-CNconditioned media; iPSC-CN lysates; AD brain lysates; and P301L taumouse brain cortex lysates; and cynomologus monkey brain lysates. Thedata show that the above-described humanized IPN002 variants arereactive with tau in a variety of samples.

A subset of the above-described humanized IPN002 variants was tested forthe ability to reduce eTau-induced neuronal hyperactivity. As shown inFIG. 19B, parental IPN002, and variants VH2/Vk1, VH2/Vk2, and VH2/Vk3blocked eTau induced hyperactivity.

Example 9: Testing the Immunogenicity of Humanized IPN002 Variants

Humanized anti-tau antibody was assessed for immunogenic potential. AnEpiScreen™ assay was used. See, e.g., Jones et al. (2004) J. InterferonCytokine Res. 24:560; and Jones et al. (2005) J. Thromb. Haemost. 3:991.Time course T cell assays were performed using CD8⁺-depleted peripheralblood mononuclear cells (PBMC); and T cell proliferation was measured byincorporation of [³H]-thymidine at various time points after addition oftest antibody samples.

PBMC were isolated from healthy community donor buffy coats (e.g., fromblood drawn within 24 hours of testing). T cell responses to a testantibody (e.g., a humanized IPN002 variant) were compared to a clinicalstandard antibody.

Purified test antibody (humanized IPN002 variant) was added to PBMCcultures in vitro to a final concentration of 50 μg/ml in culturemedium, to generate a test sample. A clinical antibody control (positivecontrol), and a culture medium-only control (unstimulated control), wereincluded as control samples. Test samples (PBMC plus test antibody), andcontrol samples, were incubated for 8 days at 37° C. with 5% CO₂. Ondays 5, 6, 7, and 7, the cells in the test and control samples weresuspended and transferred to wells of a multi-well culture plate. Thetest and control samples were pulsed with 0.75 μCi [³H]-thymidine andincubated for a further 18 hours before harvesting onto filter mats.Counts per minute (cpm) for each well were determined usingscintillation counting.

For proliferation assays, a threshold of an SI equal to or greater than2 was used, where samples inducing a proliferative response above thisthreshold were considered positive. SI (Stimulation Index) is the meantest sample counts divided by the mean of the unstimulated control.

The data are shown in FIGS. 21A-C. Healthy donor T cell proliferationresponses to a humanized IPN002 test antibody. PBMC from bulk cultureswere sampled an assessed for proliferation on days 5, 6, 7, and 8 afterincubation with the test samples. Proliferation responses with an SI≥2.0(p<0.05), indicated by the dashed horizontal line, that were significant(p<0.05) using an unpaired, two sample student's t test were consideredpositive.

As shown in FIG. 21A, a test fully humanized IPN002 antibody had lowimmunogenic potential (below the SI threshold of 2.0). FIG. 21B showsresults with a reference chimeric antibody, where the reference chimericantibody has IPN002 murine heavy and light chain variable regions andhuman IgG4 constant region; and FIG. 21C shows results with animmunogenic clinical control humanized A33 antibody.

Example 10: IPN002 Reduces the Level of Phosphorylated Tau In Vivo

The effect of IPN002 administration on the level of Tau that isphosphorylated at amino acids 202 and 205 was assessed.

The P301L mouse model was used. P301L mice are transgenic for a form ofhuman tau having a P301L mutation; P301L mice are models for humantauopathy. See, e.g., Gotz et al. (2001) J. Biol. Chem. 276:529.

P301L mice (3-4 months old) were treated with: 1) control IgG; 2) PHF1anti-phosphorylated Tau antibody; or 3) IPN002. IgG control and IPN002antibodies were injected intraperitoneally at a concentration of 10mg/kg for 4 weeks; then at 20 mg/kg for a further 4 weeks. PHF1 wasadministered at 10 mg/kg for the entire 8-week course. On day 60 afterthe beginning of the antibody treatment regimen, the level ofphosphorylated Tau was measured in the hippocampus. The data aredepicted in FIG. 22.

Tau that is phosphorylated at amino acids 202 and 205 is referred to as“AT8.” As shown in FIG. 22, treatment with IPN002 resulted in astatistically significant decrease in insoluble phospho-Tau (AT8) asassessed by ELISA (left panel), and trending toward a decrease asassessed by Western blot analysis (right panel) compared to IgG controltreatment. PHF1 treatment showed a trend toward a decrease in insolubleAT8, in support of findings by Chai et al. ((2011) J. Biol. Chem.286:34457) and Boutajangout et al. ((2011) J. Neurochem. 118:658).

Example 11: IPN002 Reduces Free Tau Levels in Both ISF and CSF

The effect of IPN002 administration on levels of free tau in CSF andinterstitial fluid (ISF) was determined. P301L mice were treated asdescribed in Example 10. The level of free tau present in ISF that isnot bound to IPN002 was determined using IPN001. As shown in FIG. 23,IPN002 treatment reduced free Tau levels (not bound to IPN002) (leftpanel) in ISF in P301L mice treated with IPN002.

To determine whether IPN002 reduces free Tau levels in CSF to the sameextent as it does in ISF, P301L mice were treated as described inExample 10, and the effect of IPN002 treatment on the level of free tau(not bound to IPN002) in the CSF of the treated mice was determined. Theresults are shown in FIG. 24.

In the left panel of FIG. 24, the levels of free tau (unbound to IPN002;referred to as “Free tau (of IPN002)”) in CSF of untreated, controlIgG-treated, PHF1-treated, and IPN002-treated mice, are shown. As shownin the right panel of FIG. 24, free tau levels in CSF is comparable tofree tau levels in ISF of IPN002-treated mice, demonstrating that ISFtau analysis correlates well with the more clinically relevant material,CSF.

Example 12: Anti-Tau Antibody Reduces eTau-Mediated NeuronalHyperactivity

Electrophysiological analyses were carried out as described in Example5. The effect of IPN002 on e-Tau-induced neuronal hyperactivity wasassessed.

As shown in FIG. 25, IPN002 reduces eTau-mediated neuronalhyperactivity.

Example 13: Tau Fragments are Present in CSF Obtained from Individualswith Likely Chronic Traumatic Encephalopathy (CTE)

CSF samples were obtained from former National Football League linemen,who exhibited behavioral/cognitive deficits, and who were consideredlikely to have CTE. The CSF samples were assayed for the presence ofeTau fragments. eTau fragments were affinity isolated from pooled CSFfrom healthy individuals and individuals with likely CTE. The isolatedeTau fragments were separated using polyacrylamide gel electrophoresis;and the separated fragments were transferred to a membrane. The membranewas probed with IPN001. The results, presented in FIG. 26, show that Taufragments are present in CSF obtained from individuals with likely CTE.

Example 14: Binding of a Humanized Variant of IPN002 to Synthetic TauPeptides

The binding of a humanized variant of IPN002 (“hu-IPN002”) to syntheticbiotinylated Tau Peptide 1 and 2 was tested in both solid phase andsolution phase assays.

The amino acid sequences of Peptide 1 and Peptide 2 are as follows:

Peptide 1 (Tau amino acids 13-24): (SEQ ID NO: 49) DHAGTYGLGDRK;Peptide 2 (Tau amino acids 15-44): (SEQ ID NO: 50)AGTYGLGDRKDQGGYTMHQDQEGDTDAGLK.

Antibody or biotinylated peptide was diluted in phosphate-bufferedsaline. The final concentrations were as follows: 1 μg/ml hu-IPN002; 1μg/ml rTau383 (full-length recombinant Tau); 5 μg/ml (biotinylatedPeptide 1); and 5 μg/ml (biotinylated Peptide 2). 100 μl 0.1% casein inPBS was added to wells of a multi-well plate. 150 μl of the 1 μg/mlsolution of hu-IPN002 was added. Serial dilutions were made. 100 μlbiotin-peptides were added to the wells containing serially dilutedantibody. The multi-well plate was incubated for one hour at roomtemperature. After the incubation period, wells were washed 5 times witha solution of 0.05% Tween 20 in PBS; followed by 2 washes with PBS.

Streptavidin-horse radish peroxidase (HRP)-conjugated secondary antibodywas added to wells, and the plates were incubated at room temperaturefor 1 hour. After the incubation period, wells were washed 5 times witha solution of 0.05% Tween 20 in PBS; followed by 2 washes with PBS.

HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added, andincubated for 1-15 minutes. The reaction was stopped by addition of 100μl 1 N sulfuric acid. Absorbance at 450 nm was read.

The results are shown in FIGS. 27 and 28, and are summarized in Table 6.

TABLE 6 Hu-IPN002 kD [M] Hu-IPN002 kD [M] Hu-IPN002 kD [M]w/biotin-rTau383 w/biotin-Peptide 1 w/biotin-Peptide 2 solid phase1.071E−10 1.062E−10 2.777E−10 solution phase 1.135E−10 1.364E−103.698E−10

FIG. 29 depicts binding of hu-IPN002 to full-length recombinant Tau(rTau-383) and phosphatase-activating domain (PAD) peptide (tau aminoacids 2-28; AEPRQEFEVMEDHAGTY; SEQ ID NO:80). As shown in FIG. 29,hu-IPN002 does not bind to PAD peptide.

FIG. 30 shows that non-biotinylated Tau Peptide 1 (Tau amino acids13-24) and non-biotinylated Peptide 2 (Tau amino acids 15-44) competewith biotinylated forms of Tau Peptide 1 and Tau Peptide 2 for bindingto hu-IPN002. These data show that the binding of Tau Peptide 1 and TauPeptide 2 to hu-IPN002 is specific and not due to the addition of thebiotin.

Example 15: In Vivo Effect of IPN002 on Pathology

In this study, the effect of a therapeutic intervention on the reducedmobility and Tau pathology in a transgenic mouse model of tauopathy(hTau.P301L-Tg) was investigated. In these mice, a clinical mutant ofhuman Tau (P301L) is expressed under control of the murine Thy1 promoter(neuron-specific expression). Behavior (beam walk) was measured at 7, 8,8.5 and 9 months of age as well as the day before study termination.End-points of the study included: (1) survival; (2) behavior: beam walkperformance and clasping score; (3) biochemistry: pan-Tau in totalhomogenate, soluble and insoluble brain stem fractions; and (4)biomarkers: AT8 in total homogenate and insoluble brainstem fractions.

Materials and Methods

P301L mice (3-4 months old) were treated with: 1) control IgG; 2) PHF1anti-phosphorylated Tau antibody; or 3) IPN002. IgG control and IPN002antibodies were injected intraperitoneally at a concentration of 20mg/kg once a week for 6 months. PHF1 was administered at 10 mg/kg for 6months. PHF1 is a mouse monoclonal antibody that recognizes an epitopethat includes phospho-Ser³⁹⁶ and phospho-Ser⁴⁰⁴. Santacruz et al. (2005)Science 309:476.

Body Weights and Clasping Behavior

Body weights were determined weekly during treatment and at sacrifice.Body weight and clasping score were recorded weekly as of study start upto the age of 7 months. From the age of 7 months onwards, mice weremonitored twice a week for mobility in home cage, weight loss and firstsigns of clasping of the hind and fore limbs. Once clasping signs werepresent, body weight was determined daily and clasping behavior wasscored until moment of ‘premature sacrifice’. To score claspingbehavior, mice were kept approximately 1.5 cm above their tail base forabout ten seconds. Clasping was scored for each limb separately using a4-point rating scale. Preliminary sacrificed mice were ascribed amaximum score. Forelimb scores were primarily used as supportingevidence for sacrifice decisions. The latter were made on the combinedobservations of clasping, weight loss and mobility in the home cage andaccording to predefined criteria. Left and right hind limb scoresunified in one clasping score were used for evaluation of claspingevolution throughout treatment and assessment of group differences atstudy termination. Mice that died prematurely from epilepsy are excludedfrom the analysis. Statistical analysis were performed using 2-way ANOVAwith repeated measures followed by Bonferroni post-hoc testing, andusing Student's T-test on the group averages of the last clasping scoresbefore sacrifice, respectively.

Beam Walk

The beam walk test was performed with the baseline group and at 7, 8,8.5, and 9 months of age, as well as the day before study termination,with the treatment groups, to determine motor dysfunction and motorlearning. The ability of a given mouse to balance on the beam and thetime that is needed to walk over a distance of 1 meter is a measure oftheir balance, coordination, physical condition and motor-planning. Acopper beam with a circular diameter of 12 mm was placed under an angleof 30°. The start area of the beam was illuminated with a desk lamp,while an indoor escape platform was placed at the end. For the initialtraining trials, a wider beam was used to train mice to balance and walkto the platform. After the training trials, the latency of the mice wastimed over a distance of 1 m. In addition, by gait observations, thefirst symptoms of motor dysfunction (foot slips and belly dragging) weredetermined. Statistical analysis of latency scores were performed using2-way ANOVA followed by Bonferroni post-hoc testing.

Results Clasping Behavior and Beam Walk

The effect of IPN002 on clasping behavior is depicted in FIG. 31. Asshown in FIG. 31, IPN002 treatment reduced the clasping score, comparedto control IgG. The effect of IPN002 on motor function, as assessed bythe beam walk, is depicted in FIG. 32. As shown in FIG. 32, IPN002treatment significantly reduced the average latency (and thus improvedmotor function), compared to control IgG. Thus, IPN002 treatmentsignificantly reduces the motor deficit in P301L tau transgenic mice.Using the same statistical method, the results with PHF1 treatment didnot reach significance.

Tau Levels

The level of free tau (tau unbound to IPN002) in the CSF of P301L micefollowing treatment with control IgG, PHF1, or IPN002 was assessed. Thelevel of free tau present in CSF that is not bound to IPN002 wasdetermined using IPN001. The data are shown in FIG. 33. As shown in FIG.33, IPN002 treatment reduced free tau (unbound to IPN002) levels by 96%,compared to the levels in mice treated with control IgG.

Example 16: Effect of IPN002 on eTau-Induced Neuronal HyperexcitabilityMaterials and Methods

Whole cell patch clamp recording was conducted on human primary corticalcultures. Neurons were perfused (2 ml/min) with artificial cerebralspinal fluid containing (mM): NaCl (140), KCl (2.5), MgCl₂ (2) CaCl₂(2), Hepes (10), D-Glucose (10), sucrose (20), adjusted pH=7.4 mOsm=310.Recordings were made using pClamp-10.3 data acquisition software(Molecular Devices) and MultiClamp 700B amplifier (Axon Instrument;Foster City Calif.). Puff application of: 1) eTau1a (amino acids 2-166);2) phosphorylated tau or eTau with inhibitors; 3) control IgG; or 4)anti-tau antibodies, PHF1, IPN001, or Dako (polyclonal anti-C-terminaltau) was performed using MiniSquirt micro-perfusion system (AutoMate,Berkeley, Calif.). Off-line data analysis used Clampfit 10.2 analysissoftware (Molecular Devices). Recordings were conducted at 34-37° C.

Results

The data are shown in FIGS. 34 and 35. As shown in FIG. 34, IPN002reduced eTau1a-induced neuronal hyperactivity. Neither control IgG nor“Dako” (anti-C-terminal polyclonal Ab) effected a significant reductionin neuronal hyperactivity. PHF1 did not reduce eTau1a-induced neuronalhyperactivity.

The effect of eTau1a on neuronal hyperactivity was compared to theeffect of full-length, PHF1-reactive, phosphorylated Tau (phospho-Tau)on neuronal hyperactivity. As shown in FIG. 35, middle panel,full-length, PHF1-reactive, phospho-Tau did not induce neuronalhyperactivity in 2 of the 3 cells tested; the third cell tested showedonly a small degree of neuronal hyperactivity, compared to baseline (toppanel). In contrast, eTau1a induced neuronal hyperactivity in all 3cells tested (bottom panel). These data are depicted graphically in FIG.36.

Example 17: Effect of IPN002 on Aβ Levels Materials and Methods

Aβ Secretion from iPSC-Derived Cortical Neurons

iPSC-derived cortical neurons (iPSC-CN) were cultured in vitro. TheiPSCs were generated from healthy individuals; individuals with familialAD with a mutation in a presenilin protein (PSEN1); an individual withfamilial AD with a mutation in a presenilin protein (PSEN2); and anindividual with sporadic AD (sAD). After approximately ˜55-60 day inculture, iPSC-CN were sorted based on L1-CAM (CD171) expression toenrich for mature cortical neurons; the sorted cells were grown inco-culture with normal human astrocytes for 30 days. After co-culturingfor 30 days, iPSC-CN were treated for an additional 25 days with variousconcentrations of beta-site amyloid precursor protein cleaving enzyme(BACE) inhibitor, control IgG, or IPN002, with 2×/week media changes.Conditioned media was collected and tested in Millipore High SensitivityHuman Amyloid-beta 40 and Amyloid-beta 42 ELISAs, to detect Aβ₁₋₄₀(“Aβ40”) and Aβ₁₋₄₂ (“A42”). Aβ40 and Aβ42 are cleavage products ofamyloid precursor protein; senile plaques contain both Aβ42 and Aβ40.

Aβ Secretion from Primary Human Cortical Neurons

Human fetal cerebral cortical tissue was obtained by Advanced BioscienceResources (Alameda, Calif.) and complied with federal guidelines forfetal research and with the Uniform Anatomical Gift Act. The tissue wasrinsed in Hank's buffered saline solution (Cellgro) and triturated inthe presence of 1 mg/ml DNase (EMD) and passed through a 100 mm cellstrainer. After centrifugation, the pellet was resuspended in 0.05%trypsin/EDTA (Invitrogen) for 20 min at 37° C. Trypsin was inactivatedby adding an equal volume of media containing 10% fetal bovine serum(FBS) and sample gently triturated again in presence of DNase. Aftercentrifugation, cells were resuspended in plating media (Neurobasalcontaining B27, Invitrogen) and counted. Cells were plated, cultured for5 weeks and fresh media added containing antibodies at the statedconcentrations for 10 days with media changes every 3-4 days withconditioned media collected after 10 days of treatment. Conditionedmedia was spun at 15,000 rpm for 15 minutes prior to processing for Aβ40and Aβ42 ELISA analysis, as described above.

Results

The results are shown in FIGS. 37-39.

As shown in FIG. 37, treatment of iPSC-CN with IPN002 reduced the levelsof Aβ40 and Aβ42 secreted by all iPSC-CN, whereas control IgG did notreduce Aβ40 or Aβ42 secretion.

FIG. 38 shows dose-dependent effects of various antibodies on the amountof Aβ40 secreted by primary human cortical neurons. As shown in FIG. 38,incubation of primary human cortical neurons with 10 μg/ml or 30 μg/mlIPN001, IPN002, or hu-IPN002 (humanized variant of IPN002) reduced theamount of Aβ40 secreted. Neither control IgG nor PHF1 had anysignificant effect on the amount of Aβ40 secreted.

FIG. 39 shows dose-dependent effects of various antibodies on the amountof Aβ42 secreted by primary human cortical neurons. As shown in FIG. 39,incubation of primary human cortical neurons with 10 μg/ml or 30 μg/mlIPN001, IPN002, or hu-IPN002 reduced the amount of Aβ42 secreted.Neither control IgG nor PHF1 had any significant effect on the amount ofAβ42 secreted.

Example 18: Epitope Mapping of a Humanized Variant of IPN002 Materialsand Methods

Antibody or biotinylated peptide was diluted in phosphate-bufferedsaline (PBS). The final concentrations were as follows: 1 μg/mlhumanized variant of IPN002 (hu-IPN002); 1 μg/ml rTau383 (full-lengthrecombinant Tau); 5 μg/ml biotinylated Peptides. 100 μl 0.1% casein inPBS was added to wells of a multi-well plate. 150 μl of the 1 μg/mlsolution of hu-IPN002 was added. Serial dilutions of hu-IPN002 weremade, and were coated onto wells of a multi-well plate. 100 μlbiotin-peptides or biotin-full-length Tau were added to the wellscontaining serially diluted antibody. The multi-well plate was incubatedfor one hour at room temperature. After the incubation period, wellswere washed 5 times with a solution of 0.05% Tween 20 in PBS; followedby 2 washes with PBS.

Streptavidin-horse radish peroxidase (HRP)-conjugated secondary antibodywas added to wells, and the plates were incubated at room temperaturefor 1 hour. After the incubation period, wells were washed 5 times witha solution of 0.05% Tween 20 in PBS; followed by 2 washes with PBS.

HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added, andincubated for 1-15 minutes. The reaction was stopped by addition of 100μl 1 N sulfuric acid. Absorbance at 450 nm was read.

Results

The data are shown in FIG. 40. The data presented in FIG. 40 show thatbiotin-Tau 13-24, biotin-Tau 15-24, biotin-Tau15-44, and biotin-phosphoTau 15-24 (with a phosphorylated Tyr corresponding to amino acid 18 offull-length Tau), and full-length Tau (rTau383) all bound efficiently tohu-IPN002. Thus, hu-IPN002 binds an epitope within Tau amino acids15-24, regardless of the phosphorylation status of Tyr-18.

Example 19: Antibody Binding to Tau in CSF

Binding of IPN002, PHF1, an antibody specific for a linearphosphorylated epitope, and control antibody, to tau present in CSF wasassessed.

A binding assay to identify antibodies that bind tau present in CSF wasdeveloped. The assay is depicted schematically in FIG. 41. Control IgG,polyclonal antibody specific for a linear phosphorylated epitope(“polyAb-C-terminal tau”), PHF1, or IPN002 was coated on wells of amulti-well plate. Antibody was diluted in phosphate-buffered saline(PBS). The final concentrations were as follows: 5 μg/ml IPN002, IgG,PHF1, or polyAb-C-terminal tau. 100 μl 0.1% casein in PBS was added towells of a multi-well plate. 100 μl human CSF was added and incubatedfor 1 hour at RT. 100 μl biotin-BT2+biotin-HT7 were added to the wells.BT2 is a mouse monoclonal antibody that binds an epitope within aminoacids 194-198 of human tau. HT7 is a mouse monoclonal antibody thatbinds an epitope within amino acids 159-163 of human tau. The multi-wellplate was incubated for one hour at room temperature. After theincubation period, wells were washed 5 times with a solution of 0.05%Tween 20 in PBS; followed by 2 washes with PBS.

Streptavidin-horse radish peroxidase (HRP)-conjugated secondary antibodywas added to wells, and the plates were incubated at room temperaturefor 1 hour. After the incubation period, wells were washed 5 times witha solution of 0.05% Tween 20 in PBS; followed by 2 washes with PBS. HRPsubstrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added, and incubatedfor 1-15 minutes. The reaction was stopped by addition of 100 μl 1 Nsulfuric acid. Absorbance at 450 nm was read.

The assay was quantitated using known amounts of full-length phospho-tauor known amounts of eTau1a (amino acids 2-166 of tau). As shown in FIG.41, lower panel, the assay was carried out using full-length tau (lowerleft panel) or eTau-1a (lower right panel), in concentrations of 0ng/ml, 0.16 ng/ml, 0.8 ng/ml, 4 ng/ml, 20 ng/ml, and 100 ng/ml. As shownin FIG. 41, lower left panel, IPN002, polyAb-C-terminal tau, and PHF1bind full-length tau. As shown in FIG. 41, lower right panel, onlyIPN002 binds eTau1a.

The above-described assay was carried out to test binding ofpolyAb-C-terminal tau, PHF1, and IPN002 to tau present in human CSFfrom: 1) control (healthy) patients; 2) MCI patients; 3) patients withmild AD (“mild”); 4) patients with moderate AD (“moderate”); andpatients with severe AD. The results are shown in FIG. 42. As shown inFIG. 42, tau present in CSF is bound by IPN002, but not by pAb-taulinear epitope or by PHF1. The data show that: 1) N-terminal Taufragments are present in CSF; 2) full-length tau was detected in CSF;and 3) no C-terminal tau fragments that include BT2 and HT7 epitopeswere detected in CSF.

Example 20: In Vivo Effects of IPN002

Results were obtained from a six month tau antibody efficacy study inP301L tau transgenic mice. It was found that IPN002 globally reducesdisease progression, as exemplified by dramatically lowered free taulevels in the CSF, reduced levels of a protein marker of astrogliosis,improvement in tau pathology across multiple brain regions andphosphotau epitopes, and improved behavioral/functional read outs.IPN002 performed as well or better than the anti-tau antibody PHF1.Finally, in vivo confirmation was obtained for a novel secreted taumechanism-of-action: positive feedback regulation of amyloid-betalevels. This study clearly distinguished the ability of the eTauantibody, IPN002, compared to PHF1, to modulate amyloid beta levels.

Materials and Methods Animal Studies

The P301L transgenic mouse model was used. The P301L transgenic mouseincludes a murine Thy1 promoter (neuron-specific expression) expressiondriven 4R2N human tau mutated at P301L as a transgene. The P301Ltransgenic model displays an age-dependent hyperphosphorylation of tau(AT8 and AT100) in spinal cord, brainstem, midbrain and cortex. Thehyperphosphorylated tau shows conformational changes which lead to tauaggregation and mice develop neurofibrillary tangles from the age of 6months, although with a high variability of onset. Concomitant to thepathology, these mice progressively develop motoric deficits such ashind limb clasping, decreased mobility on beam walk and requirepremature sacrifice at the age range between 8-11 months. Terwel et al.(2005) Proc. Natl. Acad. Sci. USA 280:3963.

100 randomized mice were dosed i.p. weekly with antibody from 3.5 monthsof age for 6 months through 9.5 months of age. 20 mg/kg (mpk) IPN002treatment was compared to a negative control antibody, 20 mpk IgG1, andto an anti-tau antibody, 10 mpk PHF1. PHF1 is a mouse monoclonalantibody that recognizes an epitope that includes phospho-Ser³⁹⁶ andphospho-Ser⁴⁰⁴. Santacruz et al. (2005) Science 309:476. Living micewere examined for clasping deficits and on beam walk performance. Micethat rapidly progressed to end-stage disease were prematurely sacrificed(prior to 9.5 months of age) using predetermined criteria. Serum, CSF,hippocampus, cortex, mid-brain and hind brain were obtained; andhemibrains were sagittally sectioned for histopathology. Antibody levelswere measured in serum and CSF; total tau and free tau (tau not bound toIPN002; “free of IPN002” tau) were measured in CSF;biochemical/histological analyses on human and mouse tau were conducted;and mouse amyloid-beta, inflammatory and synaptic protein markers andgene expression changes in inflammatory, synaptic and neuronal activitymarkers were analyzed. All analyses were conducted in a blinded manner.

The numbers of mice dedicated to this study were 25-33 mice per studyarm and 10 for baseline. All mice reserved for this study were given arandom number by computer and allocated randomly to a treatment. Themouse groups are shown in Table 7.

TABLE 7 Group N Strain Treatment 1 10 hTau.P301L-Tg None (baselinegroup) 2 32 hTau.P301L-Tg 20 mpk mIgG1 in vehicle 3 25 hTau.P301L-Tg 10mpk PHF1 in vehicle 4 33 hTau.P301L-Tg 20 mpk IPN002 in vehicle

All animal experiments were conducted in accordance with bioethicalguidelines that are fully compliant to internationally acceptedprinciples for the care and use of laboratory animals. Table 8 providesthe treatment parameters.

TABLE 8 Administration route i.p. Dosing volume, concentration 10 ml/kg;1 mg/ml; 10 mpk/day Frequency of treatment Once per week Duration oftreatment Up to 6 months dependent on survival Allocation to treatmentgroup Randomized

Body weights were determined weekly during treatment and at sacrifice.Clasping scores were recorded weekly from 7 months of age onward. From 7months of age onward, mice were monitored twice a week for mobility incage, weight loss and first signs of clasping of the hind limbs.

Clasping Behavior.

To score clasping behavior, mice were kept by the base of their tail forten seconds. Hind limb clasping is scored using a 3-point rating scale:0. Hind limbs stretched and toes spread. 1. One hind limb partiallyretracted >50% of the time. 2. Both hind limbs partially retracted >50%of the time. 3. Both hind limbs retracted completely during >50% of thetime. Forelimb clasping is scored according to 0. Forelimbs stretchedforward and distant from body. 1. One forelimb partially retracted >50%of the time. 2. Both forelimbs partially retracted >50% of the time. 3.Both forelimbs completely retracted, immobile, muscle loss, mouse is“praying”. The mice showing severe clasping phenotype were earlysacrificed.

Beam Walk.

The beam walk was performed at 7 months, 8 months, 8.5 months, 9 months,and 8.5 months of age to determine motor dysfunction and motor learning.The ability of a mouse to balance on the beam and the time needed towalk 1 meter is a measure of its balance, coordination, physicalcondition, and motor-planning. A copper beam with a circular diameter of12 mm was placed at an angle of 30°. The start area was illuminated andan indoor escape platform was placed at the far end. After the trainingtrials, the latency of the mice was timed. In addition, foot slips andbelly dragging were counted.

The baseline group was sacrificed prior to study initiation. Mice in thestudy arms that displayed severe clasping, reduced body weight and/orbecame moribund were prematurely sacrificed (early sacrifice). Theremaining mice were sacrificed after 6 months of treatment (latesacrifice).

TABLE 9 Anaesthesia A mixture of ketamine, xylazine 2%, atropine andsaline/ isoflurane. Perfusion The thoracic cavity was accessed forperfusion via trans-cardial perfusion with ice-cold saline for 3 minutesvia the left ventricle. The right atrium was cut as an outflow route.Left Dissected into hippocampus, cortex, midbrain, cerebellum hemisphereand brainstem and rest, frozen in liquid nitrogen. Right Post-fixedovernight in PBS with 4% paraformaldehyde and hemisphere stored in PBSwith 0.1% sodium azide at 4° C. CSF Collected via incision in the neckmuscles between the skull and the first cervical vertebrae. The cisternamagna was punctured with a 26 gauge needle and 10-20 ul CSF collected,centrifuged at 10,000 x g at 4° C. and stored at −80° C. Blood/Collected via heart puncture into EDTA-tubes, centrifuged plasma at 2000x g at 4° C. for 15 minutes and stored at −70° C. Body weight Measuredat sacrifice

Free PHF1 in plasma was measured by adding appropriately diluted plasmato tau-coated plates and detecting using an ELISA with HRP-anti-mouseIgG antibody and TMB. The sensitivity of the Free PHF1 assay wasinsufficient to measure free PHF1 levels in the CSF.

Free IPN002, in plasma and in CSF, was measured by adding appropriatelydiluted plasma or CSF to tau-coated plates and detecting using an ELISAwith HRP-anti-mouse IgG antibody and TMB.

Total tau in CSF was measured using a sandwich ELISA using both coatinganti-tau antibody and detecting anti-tau antibodies that weredemonstrated to not compete with either IPN002 or PHF1.

Free tau (of IPN002) in CSF was measured using a homogenous ELISA usingtwo anti-tau antibodies to capture the tau in CSF. One of theseantibodies competes with IPN002 and therefore will not interact with thetau that is previously bound to IPN002 in the CSF.

Hippocampus and cortex was fractionated by homogenizing in 10 weightvolumes of cold TBS/Roche protease/phosphatase inhibitor cocktail. Thehomogenate was generated by spinning debris out at 10,000×g for 15minutes. BCA protein content was conducted on homogenates; allhomogenates were diluted to 1 mg/ml and corresponding fractions dilutedequivalently. A portion of the homogenate was spun 1 hour at 100,000×gat 4° C. to generate a soluble fraction (S1) and insoluble pellet. Theinsoluble pellets were resuspended in 1% Sarkosyl/Rocheprotease/phosphatase inhibitor cocktail and spun again for 1 hour at100,000×g. The Sarkosyl solubilized supernatants were labeled (P1); theSarkosyl insoluble pellet was resuspended and was labeled (P2). Thehindbrains were similarly fractionated, except high salt (0.85M NaCl)followed by 1% Sarkosyl was used to solubilize the P1 pellets.

The human tau homogenous ELISA specifically reports on human tau and wasused to determine human tau levels in the homogenate, S1, P1 and P2fractions in hippocampus, cortex and hindbrain.

The human AT8 homogenous ELISA specifically reports on human p202/205tau and was used to determine human AT8 levels in the homogenate, S1, P1and P2 fractions in hippocampus, cortex and hindbrain.

HT7, IPN001, Dako polyclonal-tau, AT8, AT100, anti-p262, anti-p396 andAD2, GFAP, Iba1, synapsin SDS-PAGE Western blots were conducted onhippocampus, cortex and/or hindbrain fractions (Homogenate, S1, P1and/or P2). One or more lysate controls were run on each gel to ensureproper normalization between gels. All analyzed bands were normalized toβ-actin in the corresponding homogenate sample. Antibodies and theirtargets are shown in Table 10.

TABLE 10 Antibody Target IPN001 Tau and eTau HT7 Human tau Dakopolyclonal tau Human and mouse tau AT8 Human and mouse p202/205(phosphorylated at Ser202 and Thr205) tau AT100 Human and mouse p212(phosphorylated at Ser212) tau Anti-p262 Human p262 (phosphorylated atSer262) tau AD2 Human and mouse p396 (phosphorylated at Ser396) tau p396Human and mouse p396 (phosphorylated at Ser396) tau glial fibrillaryacidic Mouse GFAP on astrocytes protein (GFAP) ionized calcium bindingMouse Iba1 on microglia adaptor molecule 1 (Iba1) Synapsin Mousesynapsin (synaptic protein)

For histopathology, 6/32 randomized, sagittally sectioned hemibrainswere stained for AT8 and AT100; signals were developed with DAB. Boththe Subthalamic nucleus annex zona incerta (bregma 2.08-1.12) and theInterposed nucleus of the cerebellum (anterior and posterior part) annexlateral cerebellar nucleus (IntA/P/LAT; bregma 2.28-1.32) werequantified (blinded).

Mouse amyloid-beta ELISAs (both Aβ40 and Aβ42) were conducted forspecific detection of Aβ40 and Aβ42 in mouse brain homogenates. Themouse Aβ42 ELISA was not sensitive enough to reliably detect levels inmouse brain homogenates. The mouse Aβ40 ELISA detected mouse Aβ40 levelswell within the linear range of the assay. The mouse Aβ40 ELISA was usedto determine Aβ40 levels in all cohort homogenates and soluble (S1)fractions.

Taqman analysis was conducted on markers of inflammation, synapticmarkers and markers of neuronal activity. Table 11 lists the markers.

TABLE 11 Gene Function APP Amyloid precursor protein: mutated/causal insome fAD patients Human Tau MTB protein; aggregates in AD Arc Neuronalactivity Synapsin Synaptic protein Synaptophysin Synaptic proteinCalbindin Synaptic protein Neuropeptide Y Neuronal activity Cox2Inflammation GFAP Inflammation-astrocyte Iba1 Inflammation-microgliaIL-1b Inflammation P67phox Inflammation Pg91phox Inflammation PBR1Inflammation TNFa Inflammation

Statistics

Clasping and Beam walk Longitudinal statistical analysis was conductedby an independent statistician. In brief, the slope of the curve ofdecline was determined for each mouse both for clasping deficit and beamwalk and these coefficients used to determine if significant differencesexisted between groups. Multiple methods were used to determine theslope of the curves and significance. For clasping these includedLongitudinal Complete Case Analysis, Joint Model Adjusting for MissingData and Bayesian Ordered Longitudinal Analysis. For Beam walk theseincluded Longitudinal Complete Case Analysis, Joint Model Adjusting forMissing Data, Longitudinal Analysis using 30 Second Values, BayesianLongitudinal Analysis with Missing Values and Alive but Non-crossAnalysis.

Statistical analysis to determine significance for biochemistry,histology and gene expression was conducted on whole cohort, earlysacrifice and late sacrifice cohorts using one-way ANOVA followed byDunnett's post-hoc analysis. The t-test was also conducted for diseaseprogression (3.5 month baseline vs 9.5 month IgG treated arms) as acomparator to significance derived from one-way ANOVA analysis. WhenPHF1 or IPN002 appeared to be greatly trending toward changes but notreflecting this by one-way ANOVA analysis, t-test comparing IgG to PHF1or IPN002 was conducted solely to determine if trending patterns wereemerging for that endpoint without reaching one-way ANOVA significance.

To determine which of the 116 endpoints best correlate with each other,a matrix correlation was conducted; and the highest correlators wererank ordered based on a combination of their r² values and p values.

Results Antibody Levels and Target Engagement

Plasma and CSF were obtained prior to sacrifice. The levels of FreeIPN002 and Free PHF1 in plasma, and Free IPN002 in the CSF, weredetermined. The Free PHF1 assay was not sensitive enough to measure FreePHF1 levels in the CSF. Sufficient CSF was also obtained to determinelevels of both Total Tau and Free Tau (free of IPN002).

Plasma Free IPN002 and Free PHF1 Levels

P301L Tau transgenic mice were treated with 20 mpk IgG, 20 mpk IPN002 or10 mpk PHF1. These dosages were chosen based on results from the TargetEngagement #1 study. Summary data are shown in Table 12.

TABLE 12 Target Engagement Study #1 Efficacy Study Average Free AverageFree Antibody levels Antibody levels Antibody Dose administered (Plasma)Dose administered (Plasma) IPN002 10 mpk for 4 wks −> 20 0.65 μM 20 mpkfor 26 weeks  0.9 −/+ 0.26 μM mpk for 4 wks PHF1 10 mpk for 8 wks 0.65μM 10 mpk for 26 weeks 0.55 −/+ 0.05 μM

It was found in Target Engagement #1 that 10 mpk IPN002 for 4 weeksfollowed by 20 mpk IPN002 for an additional 4 weeks resulted, onaverage, in 0.65 μM Free IPN002 in the plasma. The same concentration,0.65 μM Free PHF1, was obtained with constant 10 mpk PHF1 throughout the8 weeks. In the current study, as shown in Table 12, the average plasmaconcentrations were 0.55 μM Free PHF1 and 0.9 μM IPN002. Thus, theaverage levels for Free IPN002 were higher than Free PHF1 in plasma.

CSF Free IPN002 Levels

On average, 0.9 nM Free IPN002 was present in the CSF of IPN002-treatedP301L tau mice, as shown in Table 13. This translates to 0.1% FreeIPN002 in CSF:plasma; i.e., the concentration of IPN002 in the CSF was0.1% of the concentration of IPN002 in plasma (0.9 nM in CSF; 0.9 μM inplasma). 0.1% antibody in CSF is consistent with percentages determinedfor other antibodies in accessing the brain and draining into CSFsubsequent to peripheral administration. The Free IPN002 assay, however,only reports on IPN002 that is not bound to tau. Tau is bound to IPN002in the CSF; thus, the 0.1% value underrepresents total IPN002 in theCSF. Calculations adding Free IPN002 levels to IPN002 levels bound totau suggested that the total IPN002 levels in CSF are ˜0.2% of plasmalevels. As noted in the Methods, the Free PHF1 assay is not sufficientlysensitive to measure CSF levels of Free PHF1.

TABLE 13 Efficacy Study Average Free Antibody Average Free AntibodyAntibody (Plasma) (CSF) Free 0.9 −/+ 0.26 μM 0.9 −/+ 0.28 nM IPN002

Total Tau in CSF

The levels of Total Tau in the CSF were measured to determine if eitherPHF1 or IPN002 altered these levels. As shown in FIG. 43, the levels oftotal tau in CSF were not significantly altered by PHF1 or IPN002treatment. This result was expected from the data obtained in the TargetEngagement Study.

FIG. 43: Total tau levels in the P301L tau transgenic mice CSF weremeasured using a tau antibody sandwich ELISA assay. As shown in FIG. 43,tau levels in the CSF significantly increased with age (compare 3 monthbaseline to mouse IgG). Neither PHF1 nor IPN002 altered total taulevels.

Free Tau (of IPN002) in CSF

The Free Tau (of IPN002) in CSF assay, as described in Methods, is anELISA in which one of the two tau antibodies competes with IPN002;therefore, the assay will not detect tau that is bound to IPN002. Thisassay indicates whether IPN002 has entered the brain and CSF and hasengaged its target (i.e., has bound to tau). If IPN002 has engaged itstarget, the signal in the assay will be lowered. This assay is specificto free tau of IPN002, and therefore does not detect Free Tau (of PHF1).As shown in FIG. 33, Free Tau (of IPN002) levels increase with diseaseprogression consistent with the data shown above. PHF1 does not affectFree Tau levels. In contrast, IPN002 treatment resulted in a 96%reduction in levels of the Free Tau (of IPN002) signal. These data showthat IPN002 has fully engaged its target, CSF tau.

Tau and PhosphoTau Biochemistry and Histology

As described in Methods, hippocampus, cortex, and hindbrain werefractionated into homogenate, soluble (Sarkosyl solubilized), andinsoluble (Sarkosyl insoluble) fractions. The fractions were analyzedfor both human and mouse tau, and for multiple phosphotau epitopes (AT8,AT100, p262, p396 and AD2) that are hyperphosphorylated in Alzheimer'sdisease brains. As shown in FIGS. 44 A-H, IPN002 treatment reduced AT8levels in the hippocampal homogenate (FIG. 44A), hippocampal S1 fraction(FIG. 44B), hippocampal P1 fraction (FIG. 44C), hippocampal P2 fraction(FIG. 44D), cortical homogenate (FIG. 44E), and cortical P1 fraction(FIG. 44G), compared to treatment with mouse IgG control antibody. Thedata in FIG. 44A-H show that IPN002 treatment reduced AT8 levels to asimilar or greater degree than PHF1 treatment. The data depicted inFIGS. 44A-H were normalized to BCA.

As shown in FIG. 45A, IPN002 treatment reduced the level of humanp396-tau in the cortex S1 fraction, compared to treatment with mouse IgGcontrol antibody. As shown in FIGS. 45B and 45C, IPN002 treatmentreduced the level of phospho-tau S262 in the cortex homogenate (FIG.45B), and in the cortex S1 fraction (FIG. 45C), compared to treatmentwith mouse IgG control antibody. As shown in FIGS. 45D and 45E,treatment with IPN002 reduced the level of mouse p396-tau in the cortexS1 fraction (FIG. 45D), and reduced the level of mouse AT100 in thecortex S1 fraction (FIG. 45E), compared to treatment with mouse IgGcontrol antibody.

Tau histopathology (AT8 and AT100) was analyzed in two distinct nucleiwithin the hindbrain, the Subthalamic nucleus annex zona incerta (STH)and the Interposed nucleus of the cerebellum (anterior and posteriorpart) annex lateral cerebellar nucleus (IntA/P/LAT). As shown in FIG.46, IPN002, in the late sacrifice mice, significantly improved AT8disease pathology. PHF1 trended toward a decrease, but did notsignificantly improve, tau histopathology. As shown in FIG. 47, IPN002treatment improved AT100 and MC1 disease pathology, compared totreatment with control IgG.

Inflammation

Both astrogliosis and microgliosis develop in models of AD andtauopathies. The role that activated astrocytes or microglia play indisease, however, is not entirely clear. If astrocytes or microglia areactivated in the P301L tau transgenic model, such activation wouldresult from mutated tau overexpression. It is hypothesized that secretedtau induces AD pathology. If secreted tau induces AD pathology, then itmight also induce glial activation. As such, it was determined whetherthe proteins that are increased in astrogliosis (GFAP) or microgliosis(Iba1) are increased in the P301L transgenic mouse model.

As shown in FIGS. 48A and 48B, GFAP protein levels are increased withdisease progression in both the hippocampus and cortex. These dataindicate either that astrocytes are activated in the hippocampus andcortex or that they have infiltrated these brain regions. IPN002treatment significantly reduced GFAP protein levels in both thehippocampus and cortex, showing that IPN002 treatment reduced thedisease-related increase in astrogliosis. PHF1 significantly reducedGFAP in the hippocampus but not in the cortex.

Iba1 protein levels, a marker for microglia, were measured in bothhippocampus and cortex. As shown in FIGS. 49A and 49B, Iba1 is notincreased with disease progression in the hippocampus but is increasedin the cortex. IPN002 treatment had no effect on disease progression ofIba1 protein levels. In contrast, PHF1 treatment did significantlyreduce Iba1 protein levels. The data suggest different mechanisms ofaction for IPN002 and PHF1.

Modulation of Amyloid-Beta Level

As shown in Example 17, treatment of iPSC-CN with IPN002 reduced thelevels of Aβ40 and Aβ42 secreted by all iPSC-CN, whereas control IgG didnot reduce Aβ40 or Aβ42 secretion. It was then determined whether IPN002also modulates Aβ levels in vivo. In the P301L mouse model, there is noover overexpression of human APP; therefore, mouse Aβ levels weremeasured. The sensitivity of the mouse Aβ42 ELISA was not sufficient tomeasure Aβ42 levels in these homogenates. The mouse Aβ40 ELISA, however,was sufficiently sensitive and was therefore used to determine mouseAβ40 levels in the homogenates and supernatant fractions. Mouse Aβ40levels increase with disease progression. The observed increase in Aβ40levels could be tau dependent and/or age dependent. As shown in FIGS.50A and 50B, IPN002 treatment lowered Ab40 levels in both the homogenate(FIG. 50A) and soluble fraction (FIG. 50B).

The data in FIGS. 50A and 50B show that tau overexpression (perhaps inconjunction with aging) drives a disease progression associated increasein Aβ40 levels. The data suggest that secreted tau is the causal factorin driving the increase in Aβ levels, which IPN002 in turn inhibits byblocking eTau function. PHF1, in contrast, a non-eTau binding antibody,has no effect on Aβ levels.

Motor Function

The effect of IPN002 treatment on motor function, as assessed by theclasping and the beam walk tests, was determined. The data are shown inFIGS. 31, 32, 51, and 52.

As shown in FIG. 31, IPN002 treatment improved clasping scores, comparedto treatment with mouse IgG control.

As shown in FIGS. 32 and 51, IPN002 treatment improved average latencyin the beam walk test (FIG. 32), and reduced the percent of mice unableto walk the beam (FIG. 51), compared to treatment with mouse IgGcontrol.

Example 21: Epitope Mapping

Peptide binding and competition assays were carried out as described inExample 18, above.

The following peptides were used:

IPIG-1: EVMEDHAGTYGLGDRK (SEQ ID NO: 81; amino acids 9-24 of tau);IPIG-2: DHAGTYGLGDRK (SEQ ID NO: 49; amino acids 13-24 of tau); IPIG-3:AGTYGLGD (SEQ ID NO: 82; amino acids 15-22 of tau); IPIG-4:AGTYGLGDRKDQGGYTMHQDQEGDTDAGLK (SEQ ID NO: 50;amino acids 15-44 of tau); PAD peptide:AEPRQEFEVMEDHAGTY (SEQ ID NO: 80; amino acids 2-18 of tau).

The results are shown in FIGS. 53-55.

FIG. 30 shows that unbiotinylated tau peptides compete with biotinylatedforms of tau. The upper panel shows competition of biotinylated Tau13-24 peptide (IPIG-2; SEQ ID NO:49) with unbiotinylated Tau 13-24 forbinding to hu-IPN002.

FIG. 52 shows that full-length tau, IPIG-1, IPIG-2, and IPIG-4 werebound by hu-IPN002. IPIG-3, which lacks resides 23 and 24, was not boundby hu-IPN002, nor was PAD. Thus, residues 23 and 24 appear to berequired for hu-IPN002 binding.

FIG. 53 shows that eTau-4 peptide binds hu-IPN002; however, PAD (tau2-18), Tau 15-22, Tau 19-28, and Tau 21-31 peptides did not bindhu-IPN002.

The data indicate that residues 15-24 appear to be necessary forhu-IPN002 binding.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-62. (canceled)
 63. A method of monitoring progression of a tauopathyin an individual, the method comprising: a) determining a first level ofa Tau polypeptide in a biological sample obtained from the individual ata first time point; b) determining a second level of a Tau polypeptidein a biological sample obtained from the individual at a second timepoint; and c) comparing the second level of Tau with the first level ofTau, wherein said determining comprises: i) contacting the biologicalsample with an antibody comprising a heavy chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO:37 and a lightchain variable region comprising an amino acid sequence set forth in SEQID NO:41; and ii) quantitating binding of the antibody to Taupolypeptide present in the sample.
 64. The method of claim 63, whereinthe biological sample is cerebrospinal fluid, blood, plasma, serum,urine, or saliva.
 65. The method of claim 63, wherein the quantitatedTau polypeptide is total Tau polypeptide.
 66. The method of claim 63,wherein the quantitated Tau polypeptide is an N-terminal fragment of afull-length Tau polypeptide.
 67. The method of claim 63, wherein thefirst time point is a time point before initiation of a treatmentregimen, and wherein the second time point is a time point afterinitiation of a treatment regimen.
 68. A method of detecting a Taupolypeptide in a living individual in vivo, the method comprising: a)administering to the individual an antibody comprising a heavy chainvariable region comprising an amino acid sequence set forth in SEQ IDNO:37 and a light chain variable region comprising an amino acidsequence set forth in SEQ ID NO:41; and b) detecting binding of theantibody to tau polypeptide in a brain tissue in the individual using animaging method.
 69. The method of claim 68, wherein the antibodycomprises a contrast agent suitable for use in the imaging method. 70.The method of claim 68, wherein the imaging method is magnetic resonanceimaging or positron emission tomography.
 71. An in vitro method ofdetecting a Tau polypeptide in a biological sample obtained from anindividual, the method comprising: a) contacting the biological samplewith an antibody competes for binding to an epitope within theN-terminal region of Tau with an antibody that comprises: i) light chaincomplementarity-determining regions (CDRs) of an antibody depicted inFIG. 1B; and heavy chain CDRs of an antibody depicted in FIG. 1A; or ii)light chain CDRs of an antibody depicted in FIG. 2B; and heavy chainCDRs of an antibody depicted in FIG. 2A; and b) detecting binding of theantibody to Tau polypeptide present in the sample.
 72. The method ofclaim 71, wherein the biological sample is blood, serum, plasma, urine,saliva, or cerebrospinal fluid.
 73. The method of claim 71, wherein theindividual is suspected of having a tauopathy, has been diagnosed ashaving a tauopathy, or has a genetic predisposition to developing atauopathy.
 74. The method of claim 71, wherein the method isquantitative.
 75. The method of claim 71, wherein the Tau polypeptidedetected is total Tau polypeptide.
 76. The method of claim 71, whereinthe Tau polypeptide detected is an N-terminal fragment of a full-lengthTau polypeptide.
 77. A method of reducing the level of Aβ₄₀ and/or Aβ₄₂in a neuronal cell and/or extracellular fluid in an individual, themethod comprising administering to the individual: a) an effectiveamount of an antibody comprising a heavy chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO:37 and a lightchain variable region comprising an amino acid sequence set forth in SEQID NO:41; or b) a pharmaceutical composition comprising the antibody.78. (canceled)
 79. (canceled)
 80. A method of determining the amount ofextracellular Tau (eTau) unbound to an anti-eTau antibody in a sample ofcerebrospinal fluid (CSF) or interstitial fluid (ISF) obtained from asubject undergoing therapy with the anti-eTau antibody, the methodcomprising: a) contacting an immobilized antibody with a sample of CSFor ISF obtained from the subject, wherein the immobilized antibodycomprises a heavy chain variable region comprising an amino acidsequence set forth in SEQ ID NO:37 and a light chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO:41, saidcontacting being under conditions suitable for binding of the unboundeTau to the immobilized antibody; and b) determining the amount of eTaubound to the immobilized antibody, wherein the amount of eTau bound tothe immobilized antibody is an indication of the amount of eTau unboundto the anti-Tau antibody in the sample.
 81. The method of claim 80,wherein the amount of eTau bound to the immobilized antibody isdetermined using a detectably labeled third antibody that does notcompete with the immobilized antibody for binding to the eTau.
 82. Themethod of claim 80, wherein the sample is cerebrospinal fluid. 83.-84.(canceled)
 85. The method of claim 80, comprising determining the levelof total tau in the sample.
 86. The method of claim 80, comprisingcomparing the level of unbound Tau in the sample to the level of totaltau in a CSF or ISF sample obtained from the individual before treatmentwith the anti-eTau antibody.
 87. (canceled)
 88. The method of claim 86,comprising adjusting the treatment regimen based on the compared level.89.-93. (canceled)
 94. A nucleic acid or nucleic acids encoding anantibody comprising a heavy chain variable region comprising an aminoacid sequence set forth in SEQ ID NO:37 and a light chain variableregion comprising an amino acid sequence set forth in SEQ ID NO:41. 95.An expression vector or expression vectors comprising the nucleic acidor nucleic acids of claim
 94. 96. A host cell comprising the expressionvector or expression vectors of claim
 95. 97. The host cell of claim 96,wherein the host cell is a HeLa cell, a CHO cell, a 293 cell, a Verocell, a NIH3T3 cell, a CS cell, a HEK cell, or a BHK cell.